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Aeromonas hydrophila is a widely distributed Gram-negative waterborne pathogen responsible for large-scale outbreaks of hemorrhagic septicemia in fish, causing severe economic losses to the global aquaculture industry. In view of few therapeutic drugs currently available for use in aquaculture and the emergence of multidrug-resistant A. hydrophila , there is an urgent need to develop new antimicrobial agents as an alternative to antibiotics. Antimicrobial peptides (AMPs) have strong antimicrobial effects and unique machanisms of action, and are not easy to induce bacterial resistance, making them an ideal alternative to antibiotics. In this study, we identified a new functional gene from marine crab Scylla paramamosain , named Spasin, which encodes a mature peptide of 240 amino acids and expressed widely in various tissues of crabs. Notably, a truncated peptide Spasin 141 – 165 derived from Spasin, showed potent and broad-spectrum antibacterial and antifungal activity at minimum inhibitory concentrations (MICs) ranged from 0.75 μM–48 μM, which is a novel AMP. It effectively killed S. aureus and A. baumannii within 2 h and 90 min, respectively. Importantly, it is not toxic to HEK-293 T, RAW 264.7, and ZF4 cell lines. Mechanistic studies showed that addition of 8 μg mL−1 lipoteichoic acid or lipopolysaccharides decreased bacterial susceptibility to Spasin 141 – 165. In addition, Spasin 141 – 165 altered the permeability of the inner and outer cell membranes, and resulted in the release of ATP and the accumulation of intracellular reactive oxygen species. Notably, Spasin 141 – 165 significantly increased the survival rate of zebrafish infected with A. hydrophila , redcued the bacterial load in the spleen, and effectively inhibited the expression of inflammation-associated factors (e.g. , IL-1β, TNF-α, etc.). Taken together, Spasin 141 – 165 is a potential antibacterial agent for the treatment of A. hydrophila infections in aquaculture and a promising antibiotic alternative. • Identification of a potent antimicrobial peptide from mud crabs. • Spasin 141 – 165 killed pathogenic microorganisms by disrupting cell membranes. • Spasin 141 – 165 significantly improved the survival of zebrafish infected with Aeromonas hydrophila. • Spasin 141 – 165 remarkably inhibited the inflammatory response in zebrafish after Aeromonas hydrophila infection. [ABSTRACT FROM AUTHOR]

Sulfate is an important chemical component in seawater, which has a profound influence on the survival, growth, physiology and biochemistry of aquaculture species. The aim of this experiment was to study the effects of sulfate concentration on survival, osmotic regulation, oxidative stress and immune inflammation of mud crab(Scylla paramamosain). The results showed that the 72-h semi-lethal concentration of the crab was 3.373 g/L (1.353–8.589 g/L). After 28 days of cultivation, the contents of Na+-K+-2Cl− cotransporter (NKCC) and Na+-K+ ATPase (NKA) in the gill tissue of the S1 group (0 g/L Na 2 SO 4) were significantly higher than those in the other three groups, and the sulfate content increased and the SOD activity decreased.Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were affected by sulfate content, and the content levels in the S3 group (1 g/L Na 2 SO 4) were significantly lower than those in the other three groups. The activities of ACP and AKP first increased and then decreased with the increase of sulfate content in prepare solution, and the activities of ACP and AKP in the S3 group (1 g/L Na 2 SO 4) were significantly higher than those in the other three groups. The absence of sulfate or high concentration of sulfate will affect the survival rate of mud crabs, and have a stressful effect on the osmotic regulation of mud crabs, inhibit oxidative stress ability, and promote inflammatory response. The purpose of this study was to explore the feasibility of sulfate-type saline-alkali water aquaculture of mud crab by studying the effect of sulfate on the mud crab under low salt conditions, and to provide theoretical basis and technical guidance for the promotion of saline-alkali water aquaculture. • Use Chemical reagents to prepare aquaculture water bodies that could satisfy the survival of euryhaline seafood. • Short-term semi-lethal concentration and safe concentration range of sulfate for mud crabs during culture. • Development and utilization of inland sulfate saline-alkali waters • Sulfate affects the change of water index • Effects of sulfate on survival, antioxidant capacity and immune inflammation of mud crabs [ABSTRACT FROM AUTHOR]

The Dmrt gene family, with conserved functions throughout evolution, contains DM domains that play roles in various physiological processes like sex determination and gonadal differentiation. The mud crab (Scylla paramamosain), a significant marine culture crab in China, exhibits sexual dimorphism and is an important subject for reproduction research. In this study, the ORFs of the SpDmrt-1 and SpDmrt-3 genes from the mud crab gonad transcriptome database were obtained and confirmed by PCR. Both genes were expressed at the highest level in the testis among various tissues and in the T2 stage testis during different gonad developmental stages. In situ hybridization results showed that both genes were expressed during spermatogenesis. Silencing one gene led to a decrease in the expression of the other. Moreover, the expressions of some gonadal development-related genes (dsx , foxl-2 , vasa , Dmrt-like , and IAG) changed significantly. Transcriptomic data indicated that SpDmrt-1 and SpDmrt-3 regulate various signaling pathways, including the Notch signaling pathway, the MAPK signaling pathway, the Hippo signaling pathway, the Calcium signaling pathway, and several other gender-related pathways, showing their involvement in male mud crab development. Furthermore, after long-term interference with SpDmrt-1 in vitro , most cells in the testis of the mud crab were at the spermatid stage, only a small amount of spermatozoa could be observed, and obvious empty plaque-like areas appeared in the tissue. Our findings provided a crucial foundation for understanding the role of the Dmrt in the process of gonadal development in crustaceans. • Two novel Dmrt genes Dmrt-1 and Dmrt-3 were identified from Scylla paramamosain. • SpDmrt-1 and SpDmrt-3 show a sex-biased expression pattern. • SpDmrt-1 and SpDmrt-3 play the functional role in male gonadal development and spermatogenesis. • SpDmrt-3 may be involved in other biological processes besides gonadal development. • Long-term interference with SpDmrt-1 in vitro restricts the formation of spermatozoa. [ABSTRACT FROM AUTHOR]

The world is rich in saline-alkali water resources, and the effective utilization of saline-alkali water resources is of great significance to the promotion of human economic development. This study investigated the effects of carbonate saline water on the immune, antioxidant, osmotic regulatory, and metabolic capacities of S. paramamosain. Bicarbonate stress resulted in damage to the hepatopancreas, as well as significantly upregulating the activities of superoxide dismutase and catalase, indicating that the antioxidant capacity increased under short-term bicarbonate stress, but then decreased. Long-term bicarbonate stress caused further oxidative damage. The activities of alkaline phosphatase and acid phosphatase also significantly increased, indicating an initially enhanced immune response of S. paramamosain to carbonate salinity stress that then declined, suggesting that the ability to mount an immune response decreased following long-term exposure. High bicarbonate levels also significantly inhibited the osmotic regulation ability of S. paramamosain , and negatively impacted metabolic indexes (e.g., glucose, total cholesterol, triglycerides, urea nitrogen, and uric acid). Three groups of metabolites were differentially upregulated and 95 were differentially downregulated, including 'organooxygen compounds', 'carboxylic acids and derivatives', 'fatty acyls', 'glycerophospholipids' and 'keto acids and derivatives'. Thus, long-term bicarbonate stress also decreased energy metabolism in S. paramamosain. Under long-term bicarbonate stress, related products of energy metabolism were significantly downregulated, and bicarbonate stress significantly inhibited energy uptake in S. paramamosain. This study analyzed the stress mechanism of bicarbonate on S. paramamosain , further enriched the culture theory of S. paramamosain , in order to provide a theoretical basis for the improvement of saline-alkaline water culture technology of S. paramamosain. [Display omitted] • Bicarbonate exposure damaged the hepatopancreas of Scylla paramamosain. • Stress of bicarbonate induced oxidative damage, decreased the immune response. • Bicarbonate stress inhibited the osmotic regulation ability of S. paramamosain. • Long-term bicarbonate stress reduced the metabolic level in S. paramamosain. [ABSTRACT FROM AUTHOR]

It is known that the marine euryhaline crab, mud crab (Scylla paramamosain) , is capable of adapting to low salinity conditions through an innate osmoregulatory system. However, the influence of nutritional conditions during low salinity adaptation has not been fully elucidated. In this study, transcriptome analysis was employed to examine whether low salinity affects the AMP-activated protein kinase (Ampk) signaling pathway, and thereby carbohydrate and lipid metabolism. Further analysis revealed that low salinity activated the expression of P-Ampk and promoted glycolysis and lipolysis, which enhances glycogen and lipid utilization. Nutritional deprivation confirmed that nutrients influenced the survival and osmotic pressure regulation of mud crab under to low salinity via the Ampk signaling pathway. During adaptation to low salinity, carbohydrates were the primary energy source in the initial 7 days, followed by lipids. Further investigation showed that pharmacological stimulation of mechanistic targets in the Ampk signaling pathway using metformin could promote the osmotic pressure regulation of mud crab, confirmed the pivotal role of this pathway in low salinity adaptation. Overall, the present study demonstrated that nutrition, metabolism and osmotic pressure regulation interactively contribute to salinity adaptation in mud crab. Specifically, low salinity induced remodeling of nutritional metabolism homeostasis and promoted glycolipid catabolism, thereby providing energy for osmotic pressure regulation in mud crabs. [Display omitted] • Nutrition metabolism is closely involved in low salinity adaptation in mud crabs. • Nutritional deprivation decreased the osmotic pressure regulation ability. • Enhanced utilization of carbohydrate and lipid was key for low salinity adaptation. • The Ampk is involved in the regulation of low salinity adaptation as a fuel sensor. [ABSTRACT FROM AUTHOR]

Soft-shell crabs are highly valued for their commercial significance and culinary appeal. To enhance production of soft-shell crabs, careful attention should be paid to the water calcium concentration in their culture. This study investigated the influences of different levels of calcium concentrations on the molting and growth of mud crab (Scylla paramamosain) within a meticulously controlled recirculating aquaculture system (RAS). We established three distinct water calcium concentration gradients (200, 300, and 450 mg/L) to comprehensively assess feeding habits, molting patterns, growth, and tissue calcium levels of mud crab. Initially, we observed a gradual reduction in water calcium concentrations with increments of crab molting events in RAS. Throughout one molting cycle, the feeding incidence of the crabs remained consistently high from 4 days to 37 days post-molt, irrespective of different calcium concentrations. Nonetheless, high water calcium levels (300 and 450 mg/L) significantly shortened molt intervals and accelerated exoskeletal calcification. Specifically, the molt intervals were 52.30 ± 2.72 d, 41.65 ± 5.24 d, and 37.70 ± 3.02 d, while the carapace hardening durations were 21.51 ± 4.33 h, 17.23 ± 3.38 h, and 13.46 ± 2.76 h at water calcium concentrations of 200, 300, and 450 mg/L, respectively. Moreover, elevated calcium concentrations also significantly enhanced weight gain of the mud crab during molting. Noteworthy, regardless of fluctuations in water calcium concentration, the condition factor of the molting crabs was approximately 63%, which could be an indicator of crab selection in soft-shell crab cultivation. Furthermore, the dynamic changes in calcium concentrations were disparate among different tissues in the post-molt stage. Gill and exoskeleton tissues exhibited an upward trend in calcium concentrations, while hemolymph and hepatopancreas tissues displayed distinct patterns of decrease followed by increase. Gill and exoskeleton tissues were identified as primary sites for calcium absorption and utilization, evidenced by a positive correlation between high calcium concentrations and increased calcium content in these tissues at 6 h and 3 d post-molt. Overall, our findings provide practical evidence for optimizing aquaculture management strategies through adjustments in water calcium concentration, thus to enhance production efficiency and profitability in soft-shell mud crab aquaculture. • Higher water calcium significantly shortened molt intervals, accelerated carapace hardnessduring molting cycles. • The condition factor of crabs at around 63% could be an indicator for crab selection in soft-shell crab cultivation. • Dynamic changes in calcium concentrations varied among different tissues in the post-molt stage. • Gill and exoskeleton tissues were identified as primary sites for calcium absorption and utilization. [ABSTRACT FROM AUTHOR]

A six-week feeding trial was carried out to investigate the impact of dietary ARA and CHOL levels, and their interactions, on the growth, lipid metabolism, and molting response of juvenile mud crabs (Scylla paramamosain) with an initial weight of 17.46 ± 0.04 g. A 3 × 3 two-factor design was adopted for 9 experimental diets with 0%, 0.75% and 1.5% ARA levels and 0%, 0.75% and 1.5% CHOL supplemental levels, respectively. The results showed that ARA and CHOL had significant interaction effects on percent weight gain and specific growth rate. Percent weight gain and specific growth rate of crabs fed 0.75% ARA diet were significantly higher than those of crabs fed 0% ARA diet, but only when dietary CHOL content was 0.75%. ARA and CHOL levels had significant interaction on crude lipid content and percentage of Oil Red O staining area in the hepatopancreas, and when 0.75% ARA was supplemented to 0.75% CHOL diet, the levels of crude lipid and percentage of Oil Red O staining area in hepatopancreas were significantly up-regulated. Moreover, 0.75% CHOL diet that was supplemented with 0.75% ARA significantly up-regulated the levels of DHA, EPA, and ARA in the hepatopancreas. Additionally, significant interactions between ARA and CHOL were found in expression levels of genes related to long-chain polyunsaturated fatty acid anabolism (Δ9 fad , elovl4 , elovl5 and elovl6), fatty acid anabolism and catabolism (aco3 , cpt1 and acc), fatty acid transport (fabp1 , fabp3 and fatp4), and lipid catabolism and anabolism (hsl , srebp1 , lpl and hl) in the hepatopancreas. 1.5% dietary ARA significantly increased the content of serum T-CHO and the activity of hepatopancreatic LCAT. The addition of 1.5% dietary CHOL significantly reduced the concentration of 20E in the serum. Furthermore, both 0.75% and 1.5% dietary ARA reduced concentrations of MIH and CHH in the serum. Significant interactions between dietary ARA and CHOL were observed in the concentrations of 20E and MIH in the serum. Dietary ARA significantly increased the expression levels of dib , e74b , e75 and ftz-f1 in the hepatopancreas. In conclusion, adding 6.02–11.06 mg/kg ARA to 1.21–1.27 mg/kg CHOL diets or adding 6.04 mg/kg ARA to 6.17–7.02 mg/kg CHOL diets both promoted cholesterol metabolism, deposition of essential fatty acids and lipids, and activated ecr -mediated molting response pathway, with these positive benefits likely resulting from a significant increase in PG. • Adding 6.04 mg/kg ARA to 6.17–7.02 mg/kg CHOL diet can promote the growth of mud crabs. • Dietary 11.37–11.60 mg/kg cholesterol reduced 20E synthesis and had a feedback regulation effect on MIH. • Adding 6.04 mg/kg ARA to 6.17–7.02 mg/kg CHOL diet enhanced molting response pathway. • The interaction of ARA and CHOL promoted the deposition of lipids and essential fatty acids. [ABSTRACT FROM AUTHOR]

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]

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]

Exosomes have been considered anti-microbial immune factors in animals (including aquatic animals). However, the relationship between exosomal proteins and immune responses during bacterial infection has not been addressed. Flotillin-1 has previously been shown to enrich plasma membrane which implicates in cellular processes, including signal transduction, membrane trafficking, and molecular sorting. In this study, Sp Flotillin-1 was cloned and characterized from mud crab (Scylla paramamosain). Sp Flotillin-1 was found to be up-regulated in the hemocytes of mud crabs after infecting with V. parahaemolyticus. RNAi knockdown of Sp Flotillin-1 showed capacity in suppressing phagocytosis and reducing the expression of antimicrobial peptides (AMPs) but increasing the ROS production in hemocytes. Furthermore, Sp Flotillin-1 densely packaged in the exosomes can regulate the phagocytosis, expression of AMPs through MAPK and NF-κB pathway, and production of ROS, eventually activating the immune response to bacterial infection in mud crabs. Taken together, the results of this study provide a new finding on the mechanism that exosomal Sp Flotillin-1 may participate in the V. parahaemolyticus infection through the regulation of phagocytosis and activation of AMP synthesis in mud crabs. • Expression of Sp Flotillin-1 was increased in mud crab hemocytes during bacterial infection. • Sp Flotillin-1 RNAi suppressed phagocytosis and AMP expression, and increased ROS production in hemocytes. • Exosomal Sp Flotillin-1 activates AMP synthesis via NF-κB and MAPK signaling pathways. [ABSTRACT FROM AUTHOR]

A 56-day feeding trial was designed to investigate the effect of different dietary lysine (Lys) levels on the growth, molting, lipid metabolism, antioxidant, and immunity of postlarva Scylla paramosain. Six diets with 1.20%, 1.46%, 1.84%, 2.06%, 2.35%, and 2.67% Lys levels (dry matter) were formulated and experimented on the postlarva Scylla paramamosain. The results showed that Scylla paramamosain fed with the 2.06% lysine obtained the highest growth performance and the highest molting frequency. Appropriate dietary Lys levels (1.73–2.03%) can improve the antioxidant capacity of Scylla paramamosain. Although unaffected by dietary Lys, the whole-body lipid content of the 1.20% Lys group was numerically higher than those of the 1.46–2.67% Lys groups. The variation in whole-body crude protein content showed a similar trend with those in the content of EAA and NEAAs, with the highest whole-body crude protein content appearing in the 2.06% Lys group. The crabs obtained the highest expression of igf , fas , EcR and fabp genes in the 2.06% Lys group. When dietary Lys levels were > 1.20%, the transcription level of immune related-genes increased. As suggested by the results of the second order polynomial regression analysis of weight gain, the Lys requirement of postlarva Scylla paramamosain is 2.03% (4.51% of dietary protein). • The optimal dietary lysine level for postlaval S.paramamosain was 2.03% (4.51% of the dietary protein). • This study indicates that appropriate dietary lysine can promote the expression of lipid metabolism genes in this species. • This study indicates that appropriate dietary lysine can promote protein synthesis in this species. • This study indicated that an adequate lysine content in diet can improve the health of postlaval S. paramamosain. [ABSTRACT FROM AUTHOR]

This study investigated the effects of different overwintering temperatures (8, 12, 16, and 20 °C) on basic nutritional composition and non-volatile flavor substances, including free amino acids and free nucleotides of edible tissues (gonad, hepatopancreas, and muscle) of female mud crab Scylla paramamosain after eight-week of overwintering. The results showed that the overwintering process and temperature did not affect the basic nutritional composition of the gonad (P > 0.05). However, with the increase in temperature, the lipid content of hepatopancreas decreased and was accompanied by a rise in moisture content (P < 0.05). In muscle, moisture content increased significantly, and crude protein content decreased significantly after overwintering at 20 °C (P < 0.05). During the overwintering process and with the increase in temperature, free amino acids and free nucleotides decreased considerably (P < 0.05). In addition, each tissue had a high content of total sweet amino acids (SAA) under low overwintering temperatures. However, the contents of total umami amino acids (UAA) and equivalent umami concentration (EUC) increased significantly, accompanied by the decrease of total bitter amino acids (BAA) with the rising temperature (P < 0.05). To sum up, overwintering under high temperature was beneficial to improve the umami characteristic and reduce the bitter characteristic, but it would cause the crabs to lose and consume proteins and lipids. However, overwintering at low temperature had higher nutrients and sweet characteristic. Therefore, overwintering at 8–12 °C is more conducive to improving the nutritional quality and flavor of S. paramamosain. • Increasing temperature significantly decreased hepatopancreas lipid content and muscle protein content. • High-temperature overwintering improved umami and reduced bitterness. • Overwintering at low temperature had higher sweet characteristic. • Overwintering at 8–12 °C enhanced S. paramamosain 's nutritional quality and flavor. [ABSTRACT FROM AUTHOR]

Abstract Mud crab (Scylla paramamosain) is an economically important crustacean species for fisheries and aquaculture industry in China and other Southeast Asian countries. In this study, the sex ratio, morphological dimorphism, and the characteristics of morphological traits between female and male S. paramamosain juveniles at three different pond-culture stages were determined. The sex ratio skewed towards female crablets as growth and development proceeded. Three growth traits exhibited significant differences between sexes when the crablets were at two months of pond-culture stage, however, the number of growth traits with significant differences increased to seven when the crablets were at three and four months of pond-culture stages. Further, significant positive correlations were detected among all the 190 trait-pairs, irrespective of the pond-culture stages of the crablets. Apart from this, the path coefficients and determination coefficients varied between sexes among the three pond-culture stages, suggesting that the growth traits have different effects on body weight (BW) of female and male crablets during different pond-culture stages. The effect of growth traits on BW in both sexes at different pond-culture stages was determined by constructing six multiple regression equations. Analysis of the width-weight relationship revealed that male crablets had positive growth allometry whereas the females had isometric growth. In addition, an obvious sexual dimorphism in cheliped size and abdomen width was observed with growth and development. A discriminant function equation was successfully constructed for sex identification based on morphological indexes, with a high accuracy of 90.4%. Highlights • The sex ratio and sexual dimorphism of Scylla paramamosain crablets were evaluated. • The morphological traits affecting body weight varied during the different developmental stages. • A sex discriminant equation based on morphological indexes was constructed, with a high accuracy of 90.4%. • The findings serve as the baseline information for prospective artificial selective breeding and farming of S. paramamosain. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor B (VEGFB), unlike other members of the VEGF family such as VEGFA, shows only limited potential in angiogenesis in mammals, but is involved in lipid metabolism, especially in transporting dietary lipids present in circulation through the vascular endothelium to peripheral tissues. However, the role of Vegfb in invertebrates such as decapod crustaceans with an open circulatory system is still poorly understood. Here, cDNA of a vegfb -like gene was cloned and characterized for the first time from a decapod crustacean, mud crab Scylla paramamosain. The mud crab vegfb -like gene encoded a polypeptide of 386 amino acids that possessed all the typical features of the Vegf family, and shared high homology with other crustacean Vegf orthologs. Relative expression levels of the vegfb -like gene were higher in metabolically active tissues such as gill, heart and muscle, and increased in ovary of female crabs as ovary matured. Knockdown of the vegfb -like gene in vivo reduced the contents of triglyceride in hemolymph and hepatopancreas, as well as total lipid in hepatopancreas. In addition, knockdown of the vegfb -like gene increased tissue expression levels of genes related to fatty acid transportation, allocation and hydrolysis, such as long-chain acyl-CoA synthetase 3/4 (acsl3/4), fatty acid transport protein 1 (fatp1), and hormone-sensitive lipase (hsl), and decreased the expression levels of the genes related to fatty acid synthesis like acetyl-CoA carboxylase (acc). These results in mud crab suggest that Vegfb might play important roles in regulating fatty acid absorption, utilization and allocation in crustaceans. • The Vegfb-like gene is first cloned and function characterized from decapod crustacean Scylla paramamosain. • The Vegfb-like potentially contributes to ovarian lipid accumulation during ovarian maturation of mud crab. • The Vegfb-like is involved in lipid accumulation and metabolism in crustacean. [ABSTRACT FROM AUTHOR]

The mud crab (Scylla paramamosain) is an important economic breeding species along the southeast coast of China. As a crustacean, molting is critical for growth and reproduction. At each molting, a new external cuticle of mud crab is inherently regenerated, but along with molting the crabs are easily susceptible to pathogenic infection that might result in a low survival rate particularly at juvenile stages. To elucidate the potential response mechanism of juvenile mud crabs infected with bacteria in the molting process, integrated transcriptomic and metabolomic techniques were applied and the hormones, associated-hormone genes or immune genes modulated by challenge with Staphylococcus aureus or Vibrio alginolyticus were analyzed. The omic analysis showed that S. aureus or V. alginolyticus infection caused decreases in steroid hormones (cortisol, cortodoxone, cortisone, deoxycorticosterone) and fatty acid-derived hormones (prostaglandin A2, Δ17–6-keto prostaglandin F1α, 13,14-Dihydro-15-keto-tetranor prostaglandin F1α, prostaglandin B2, 2,3-Dinor-11β-prostaglandin F2α, thromboxane B2, and 11-Dehydro thromboxane B2). Correspondingly, transcriptomic analysis revealed that both S. aureus and V. alginolyticus infection downregulated the expression levels of fatty acid-derived hormone synthases. In addition, both bacterial infections caused upregulation in Toll, IMD, the Jak/Stat pathway, antimicrobial peptide, and the prophenoloxidase system. The study provides a new insight into the effects of bacteria on the juvenile crab molting process in vivo that lead to the modulation of both immune-associated genes and hormones. • Bacterial infection upregulated many immune genes during the molting process. • Steroid hormones and fatty acid-derived hormones were reduced during the molting process with bacterial infection. • Cortisol were significantly reduced after infection with both S. aureus and V. alginolyticus. • Many peptide hormones of mud crab were upregulated after bacterial infection. [ABSTRACT FROM AUTHOR]

Hemolymph and midgut are important tissues in the invertebrate's immune response, ROS generated by Sp Nox plays a vital role in regulating microbiota and maintaining homeostasis. In this study, the function of Sp Nox in regulating the hemolymph and midgut homeostasis in mud crab (Scylla paramamosain) was investigated. The results showed that Sp Nox regulated the microbiota harboring the hemolymph and midgut of mud crab through generating ROS. Furthermore, the ROS (principally O 2 −) derived from Sp Nox can modulate the composition of microbiota in the hemolymph (including Bacteroides , Aeromonas , Alteromonas , and Lysobacter) and in the midgut (including Arcobacter , Aliivibrio , Hypnocyclicus , Carboxylicivirga , and Candidatus_Bacilloplasma). Apart from Sp Nox, other ROS-related genes also participate in the regulation of hemolymph microbiota at certain times. Further studies to explain the detailed mechanisms that Sp Nox regulates the microbiota in aquatic animals are promising, which can provide good theoretical support for the healthy culture of aquatic animals. • Sp Nox can change the microbial diversity and composition of hemolymph and midgut in mud crabs. • Sp Nox can modulate the microbial homeostasis through regulating ROS production. • Sp Nox might promote the homeostasis by regulating specific bacteria in hemolymph and midgut. [ABSTRACT FROM AUTHOR]

Photoperiod is one of the most important environmental cues for the organism, and it plays a critical role in regulating organisms' survival, growth, and metabolism. In the present study, the effects of photoperiod (0 L∶24 D, 6 L∶18 D, 12 L∶12 D, 18 L∶6 D, and 24 L∶0 D) on mortality, growth performance, and lipid metabolism of juvenile mud crab (Scylla paramamosain) were studied with an 8-week experiment. The results showed that the highest final weight of crabs was observed in the 18 L∶6 D (0.48 ± 0.08 g), which was significantly higher than 0 L∶24 D (0.22 ± 0.03 g) and 6 L∶18 D (0.28 ± 0.04 g) (P < 0.05). In addition, the specific growth rate (SGR) of crabs reared at 12 L∶12 D (natural photoperiod) (5.83 ± 0.26% day−1) and 18 L∶6 D (6.28 ± 0.27% day−1) were significantly higher than 0 L∶24 D (4.70 ± 0.42% day−1), 6 L∶18 D (5.16 ± 0.22% day−1) and 24 L∶0 D (5.60 ± 0.23% day−1) (P < 0.05). The survival rate of the natural photoperiod group was significantly higher than 18 L∶6 D group (69.23 ± 6.28% vs 35.90 ± 3.63%) (P < 0.05), but no significant difference between 0 L∶24 D (58.97 ± 3.63%), 6 L∶18D (66.67 ± 7.25%) and 24 L∶0 D (58.97 ± 7.25%) groups was observed (P > 0.05). Crabs exposed to 18 L∶6 D photoperiod also had higher melatonin (311.12 ± 79.53 pg mL−1 vs 168.79 ± 31.24 pg mL−1) and cortisol levels (451.37 ± 237.64 ng mL−1 vs 112.91 ± 39.32 ng mL−1) than those reared in constant light (24 L∶0 D) (P < 0.05). In addition, compared with natural photoperiod, crabs reared under constant darkness showed significantly higher TG and TC content (P < 0.05). Moreover, in constant darkness, lipogenesis-related genes such as fatty acid synthase (fas), sterol regulatory element binding protein-1 (srebp-1), and acetyl-CoA carboxylase (acc) of the crab were up-regulated, and lipolysis-related genes as carnitine palmitoyltransferase-1 (cpt-1), Acyl-CoA oxidase-3 (aco-3) were significantly down-regulated (P < 0.05). Besides, fatty acid binding protein (fabp) was also up-regulated in crabs reared under constant darkness. Thus, these results suggest that the crabs in constant darkness did not effectively utilise the lipid as an energy source. Overall, the present research confirmed that light cues are indispensable for the indoor mud crab culture system, and the natural photoperiod was recommended for mud crab during the juvenile grow-out phase in commercial operations. • Natural photoperiod (12L:12D) positively affected the survival and growth in juvenile S. paramamosain. • The SGR of crabs reared under natural photoperiod was 13.07% and 23.99% higher than those raised in 0L:24D and 6L:18D. • Constant darkness promoted the relative expression of fas , acc and srebp-1 , inhibited cpt-1 and aco-3 , and negatively affected the SGR of the mud crab. • The recommended photoperiod for growth performance of juvenile S. paramamosain is 12L:12D. [ABSTRACT FROM AUTHOR]

The effects of the overwintering temperature (8, 12, 16, and 20 °C) on survival, antioxidant capacity, and heat shock protein (HSP) gene expression in Scylla paramamosain were examined during an experimental period of eight weeks. The effects of the temperature on survival were parabolic, with the highest and lowest survival rates occurring at 16 °C and 8 °C, respectively. Histological examination of the hepatopancreas revealed that the hepatopancreas tissue structure remained intact and that cell boundaries were clearly observed at 16 °C and 20 °C. However, hepatopancreas cells in the 12 °C and 8 °C groups appeared to be vacuolated and indistinct, respectively. Furthermore, high levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and low levels of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) in the hepatopancreas were observed in the 8 °C group. Hsp60 and hsp70 gene expression were dramatically upregulated, but hsp10 , hsp40 , hsp90 , and glucose regulatory protein 78 (grp78) gene expression were downregulated at 8 °C. The terminal deoxynucleotidyl transferase deoxy-UTP nick end labeling (TUNEL) method showed that there was more DNA damage at 8 °C and 12 °C than at 16 °C. Moreover, apoptosis was significantly induced with decreasing temperature, and the 8 °C group had the highest apoptosis index. Overall, these results suggest that the overwintering temperature greatly impacts the survival of mud crabs and that these effects may be mediated through antioxidant, HSP response, and apoptosis pathways. Significantly, the optimal overwintering temperature for S. paramamosain was extrapolated to be 14.2–16.1 °C. This suggests that temperature control should be considered in the process of mud crab overwintering. • Overwintering temperature significantly affected the survival of female adult mud crab Scylla paramamosain in RAS. • The mud crab has a comparatively poor adaption to long-term cold stress. • The effects could be mediated through antioxidant, HSP response, and apoptosis pathways. • The optimal overwintering temperature for S. paramamosain was extrapolated to be 14.2–16.1 °C. [ABSTRACT FROM AUTHOR]

A 60-day culture experiment was designed to investigate the effect of dietary phosphatidylcholine/soybean lecithin (PC/SL) levels on survival, growth, body composition, antioxidant, and osmoregulation ability of early juvenile mud crab Scylla paramamosain. Six isonitrogenous and isolipidic experimental diets were formulated with five graded PC levels (0.1% (PC0.1), 0.5% (PC0.5), 1.0% (PC1.0), 2.0% (PC2.0), and 4.0% (PC4.0)) and one SL level (2.0%, SL2.0) to fed early juvenile S. paramamosain (8.70 ± 0.19 mg/crab). The results showed a significantly low survival in the PC0.1 group. Mud crabs obtained the highest growth performance in the PC2.0 and SL2.0 groups, but there was no significant difference with crabs in the PC0.5, PC1.0, and PC4.0 groups. The expression of the target of rapamycin (tor) gene was positively correlated with growth performance. The crab's whole-body crude lipid, triglyceride (TG), and total cholesterol (T-CHO) content rose significantly with increasing dietary PC levels. At the same time, the crab whole-body long-chain polyunsaturated fatty acids (lc-PUFA) proportion increased significantly, accompanied by a decrease in the proportion of C18:2n6. The expression of fatty acid synthesis-related genes decreased significantly with the increase in dietary PC levels. Dietary PC levels ≥1% significantly increased the antioxidant capacity of the mud crabs. Crabs obtained the highest total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activities in the PC1.0 group; the catalase (CAT) activity was positively correlated with dietary PC levels; significantly lower malondialdehyde (MDA) content was found when dietary PC ≥ 1%. Na+/K+-ATPase (NKA) activity increased with increasing dietary PC levels, and the expression of Na+/K+/2Cl−-cotransporter (nkcc) and nka genes were also significantly elevated. The results of the salinity challenge showed that the crabs obtained the highest survival in the PC4.0 group. In summary, the second-order polynomial regression analysis of weight gain and specific growth rate showed that early juvenile S. paramamosain obtained the highest growth at a dietary PC level of 2.60–2.62%. • The optimum dietary PC was 2.60–2.62% for juvenile Scylla paramamosain. • Dietary PC improved the retention but not the synthesis of lc-PUFA in mud crabs. • Dietary PC improved antioxidant and osmoregulatory capacity of mud crabs. [ABSTRACT FROM AUTHOR]

Two new peptides homologous to the reported antimicrobial peptide Scygonadin were characterized in Scylla paramamosain with 48.51% and 49.23% identity in the amino acid sequences, named as SCY4 and SCY5, respectively. The mRNA transcripts and proteins of the two peptides were abundant in the ejaculatory duct of male crabs, and they were transferred to the spermatheca of female crabs along with semen during mating. Immunofluorescence analysis showed that both peptides were localized in sperm cells at different developmental stages and in the acrosomal tubes of acrosomal reactions, suggesting their potential roles in protecting sperm and in the acrosomal reactions. The analysis of progesterone stimulation in vivo showed that the expression patterns of SCY4 and SCY5 were different, with SCY5 up-regulated and SCY4 down-regulated. The recombinant proteins (rSCY4 and rSCY5) were more active against Gram-positive bacteria than the Gram-negative bacteria we tested. It is noteworthy that although rSCY4 and rSCY5 had no bactericidal effect on the pathogen Vibrio alginolyticus in vitro , both proteins could obviously enhance the survival rate of crabs challenged with V. alginolyticus. In summary, SCY4 and SCY5 may play a role similar to Scygonadin in the reproductive process, thereby maintaining internal sterility during sperm transfer or in the spermatheca of female S. paramamosain. • Identification of two new genes SCY4 and SCY5 homologous to Scygonadin in mud crab. • SCY4 and SCY5 were abundant in male gonad and post-mating female spermatheca. • SCY4 and SCY5 were detected in sperm development and acrosomal reaction. • Injecting progesterone in vivo , SCY4 and SCY5 showed converse expression patterns. • rSCY4 and rSCY5 notably improved crab survival under Vibrio alginolyticus challenge. [ABSTRACT FROM AUTHOR]

Semicarbazide (SEM) is a common pollutant in the marine environment. It can cause cell mutation, carcinogenesis, and teratogenesis, threatening the health and survival of marine species. In this study, the immunotoxicity of SEM was investigated in a marine crustacean species, the mud crab Scylla paramamosain. After seven days of exposure to 0.2 μg/L, 2 μg/L, or 20 μg/L concentrations of SEM, the effects of SEM on hemocytes and immune function were analyzed in S. paramamosain. The results showed that the hemolymph color deepened and total hemocyte count (THC) was down-regulated significantly with increasing SEM concentration. Meanwhile, tissue sections showed cell infiltration, cytoplasmic vacuolization, and cell steatosis. The survival rate of hemocytes decreased after SEM treatment in vitro , which revealed that SEM showed a cytotoxic to hemocytes. Superoxide dismutase (SOD), glutathione (GSH), phenoloxidase (PO) and catalase (CAT) activity decreased with increasing SEM concentration, while the activity of glutathione peroxidase (GSH-Px) was increased compared with the control group. Similarly, intracellular ROS content increased to about 1.2-, 1.3-, and 2-fold that of the PBS group after exposure to 0.2, 2, and 20 μg/L SEM, respectively. There was a trend of significant up-regulation of p53 and caspase-3 with increasing concentrations of SEM identified by Q-PCR. Meanwhile, the hemocytes apoptosis rate were increased with the SEM concentration, which induced by caspase-3 pathway activated by SEM. The phagocytic activity of hemocytes decreased with increasing SEM concentration. The challenge experiment illustrated that SEM exposure reduced the resistance of mud crab to Vibrio alginolyticus. Our results demonstrated that SEM exposure could inhibit the immune function by causing cell damage, inducing reactive oxygen species (ROS), reducing the activity of antioxidant enzymes and inducing hemocytes apoptosis in a marine crustacean species. SEM in seawater could inhibit the immune function and threaten the survival in crustaceans. • Semicarbazide (SEM) had a cytotoxic effect on hemocytes in mud crabs. • SEM exposure induced ROS, caused DNA damage, and reduced hemocyte viability. • SEM reduced superoxide dismutase, phenoloxidase, catalase, and glutathione activity. • SEM exposure reduced the bacterial disease resistance of mud crabs. [ABSTRACT FROM AUTHOR]

Protein inhibitor of activated STAT (PIAS) plays a critical role in the feedback modulation of various signaling pathways as a negative regulator. To further understand the role of PIAS in immune and stress response in mud crab ( Scylla paramamosain ), in the present study, a full-length cDNA of PIAS from S. paramamosain (SpPIAS) was cloned for the first time, which contained an open reading frame of 2364 bp encoding a polypeptide of 788 amino acids. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze the tissue distribution of PIAS in mud crab. The results revealed that SpPIAS was expressed in hemocytes, heart, hepatopancreas, gills, stomach, intestines, muscle, connective tissue and gonad of S. paramamosain , and the higher expression levels were found in connective tissue and gonad. The expression levels of SpPIAS in hemocytes were up-regulated after challenge with Staphylococcus aureus , Vibrio harveyi , and white spot syndrome virus (WSSV) for 2–24 h, which suggested that SpPIAS was involved in the pathogen-resistant activities of mud crab. Furthermore, the RING-like domain (RLD) and Serine/Threonine rich repeat C-terminal domain (S/T) of SpPIAS were expressed by prokaryotic expression system and purified by glutathione Sepharose 4B chromatography. This study contributed to define the biological characteristics of SpPAIS and further demonstrate the critical role of SpPAIS in vivo during the pathogen infection. [ABSTRACT FROM AUTHOR]

This study was carried out to determine the effects of dietary lipid levels on growth performance, feed utilization, body composition and antioxidant parameters of juvenile mud crab ( Scylla paramamosain ). Five isonitrogenic and isoenergetic experimental diets (45% crude protein) containing 0%, 3%, 6%, 9% and 12% added oils (named L0, L3, L6, L9 and L12, respectively) were fed to triplicate groups of 20 crabs (initial weight 11.53 ± 0.52 g, carapace width 3.92 ± 0.14 cm) for 8 weeks. The results showed that the survival, final body weight, carapace width, specific growth rate and molting frequency of crabs fed diet L6 were significantly higher than those of crabs fed diet L0, L3 or L12 ( P < 0.05). The highest feed conversion ratio and the lowest protein efficiency ratio were both observed in crabs fed diet L0 ( P < 0.05). The maximum voluntary feed intake was found in crabs fed diet L3 ( P < 0.05). The lipid contents of whole body and hepatopancreas increased remarkably with the elevated dietary lipid levels ( P < 0.05). The proportions of saturated fatty acid and monounsaturated fatty acid of the hepatopancreas were significantly higher in crabs fed diet L0 ( P < 0.05). The fatty acid composition of the hepatopancreas and muscle was consistent with those in the diets. In the hepatopancreas, crabs fed diet L12 accumulated the highest concentration of malondialdehyde than other groups ( P < 0.05), and the activities of antioxidant enzymes (superoxide dismutase, glutathione S-transferase and glutathione peroxidases) were all enhanced with the increased dietary lipid levels ( P < 0.05). The results suggested that a proper dietary lipid level of 8.52%–11.63% (optimum 9.5%) could maintain solid growth performance and antioxidant capacity of juvenile mud crab S. paramamosain . [ABSTRACT FROM AUTHOR]

Janus kinase (JAK), a nonreceptor tyrosine kinase, is the JAK-signal transducer and activator of transcription (STAT) signaling pathway. In this study, RNA interference technology was used to knockdown the expression of JAK to explore the immune function of JAK in Scylla paramamosain. JAK expression was relatively high in the intestine and relatively low in the heart and hemocytes of S. paramamosain. JAK was highly expressed in all examined tissues of S. paramamosain after infection with white spot syndrome virus (WSSV) or Vibrio alginolyticus. The expression levels of Astakine , STAT , CAP , TLR , proPO , and CTL were downregulated, while MCM7 , Myosin and Relish expression was upregulated in hemocytes at 24 h after injection of JAK -dsRNA. Astakine expression was significantly downregulated within 72 h after injection of JAK -dsRNA. The total hemocyte count and hemocyte proliferation were significantly decreased after treatment with Astakine -dsRNA or JAK -dsRNA. The results showed that JAK may promote hemocyte proliferation, probably by regulating Astakine expression. The activities of phenoloxidase, superoxide dismutase, and lysozyme were decreased significantly, while production of reactive oxygen species was increased significantly in hemocytes after treatment with JAK -dsRNA. After JAK knockdown, the phagocytosis rate of WSSV or V. alginolyticus in hemocytes was increased. After WSSV challenge, the WSSV copy number in the JAK -dsRNA-treated group was higher than that in the WSSV group. After V. alginolyticus challenge, the apoptosis rate of hemocytes in the JAK -dsRNA group was higher than that in the V. alginolyticus group. The mortality rate of WSSV or V. alginolyticus -infected crabs after JAK knockdown was higher than that of the infection group. These results suggest that JAK can regulate the expression of some immune-related genes, such as Astakine, to influence the innate immune response of S. paramamosain against pathogens (WSSV or V. alginolyticus), mostly by regulating enzyme activities, hemocyte phagocytosis and apoptosis. • Total hemocyte counts and hemocyte proliferation were regulated by JAK. • Astakine expression was regulated by JAK in crabs. • JAK is involved in the immune response of S. paramamosain to pathogens. [ABSTRACT FROM AUTHOR]

It is well known that the androgenic gland is a specific endocrine organ of the male crustacean, controlling its primary and secondary sexual characteristics. The androgenic gland hormone is a key peptide hormone involved in sexual differentiation in crustaceans. In this study, a full length cDNA encoding insulin-like gland hormone from Scylla paramamosain , named Sp-IAG , was identified. The predicted protein had similar molecular organization to other insulin-like androgenic gland factors reported in the Decapoda, encoding a signal peptide, B chain, C peptide and A chain. There were two disulfide bridges between the B chain and A chain, and one disulfide in the B chain. In particular, a full genomic DNA sequence of Sp-IAG with a 5′ flanking region of 2988 bp was revealed from which many potential regulatory binding sites were first identified in S. paramamosain . The Sp-IAG gene was highly expressed in the androgenic gland and also lowly expressed in the seminal vesicle and ejaculatory duct, which was not reported in previous studies. It is noteworthy that Sp-IAG was highly expressed both in the androgenic gland and in the seminal vesicle using western blot analysis, suggesting that Sp-IAG might play a major role in these two organs. In addition, it was quantitatively detected that Sp-IAG mRNA expression was up-regulated after mating, which suggested that this protein might have an activity related to mating behavior. Eyestalk ablation can induce a high level of Sp-IAG expression, demonstrating that the Sp-IAG of S. paramamosain was negatively controlled by the X-organ–sinus gland in the eyestalk. These findings provided the basis for elucidating the mechanism of sex differentiation in crustaceans. [ABSTRACT FROM AUTHOR]

Estradiol 17 β-dehydrogenase (17E2DH) is an oxidoreductase gene involved in the metabolism of androgen and estrogen in mammals and birds, and is also associated with molting in crustaceans. However, neither the full-length estradiol 17 β-dehydrogenase nor its molecular mechanism during molting has been reported in crabs. In this study, the cDNA encoding 17-beta-dehydrogenase was cloned from the hepatopancreas of Scylla paramamosain and named Sp17E2DH. The full length Sp17E2DH consists of 922 bp with an open reading frame encoding 255 amino acids. Phylogenetic analysis showed that the predicted Sp17E2DH protein is conserved and clustered with the 17E2DH from other crustacean species. RT-qPCR and immunohistochemistry analyses indicated that the expression level of Sp17E2DH was lowest before molting (TQ), up-regulated at 3 h after molting (TH3h), and highest at 48 h after molting (TH48h) in the hepatopancreas and muscles. An RNA interference experiment showed that the mRNA expression level of Sp17E2DH was significantly down-regulated (P < 0.05) and the hardness of crab shells was significantly decreased (P < 0.05) at TH48h in the hepatopancreas and muscles after injection of Sp17E2DH -targeted dsRNA. Genes such as gastrolith protein (P = 0.045), cuticular protein 15 (P = 0.021), chitin synthase (P = 0.047) and troponin C2 (P = 0.013) were significantly suppressed at TH48h after Sp17E2DH dsRNA injection compared to the control groups, which were potentially regulated by Sp17E2DH to control shell hardness of S. paramamosain. In summary, the results suggest that Sp17E2DH plays a vital role in the molting cycle and shell hardening of the mud crab, and the dsRNA interference of Sp17E2DH may be used to prolong the marketing shelf life of live soft-shelled mud crabs. • Estradiol 17 β-dehydrogenase gene of Scylla paramamosain were cloned and characterized. • The expression level of Sp17E2DH gradually increases in hepatopancreas and muscles after molting. • The RNA interference of Sp17E2DH significantly inhibited crab shell hardness. • RNAi of Sp17E2DH significantly changed mRNA expression level of molting related genes. • RNAi of Sp17E2DH might be used to prolong the marketing shelf life of live soft-shelled mud crabs. [ABSTRACT FROM AUTHOR]

To investigate the metabolic response of mud crab Scylla paramamosain to dietary ratio of docosahexaenoic acid (DHA) to eicosapentaenoic acid (EPA), transcriptomic and lipidomic analyses were conducted on the hepatopancreas of crabs (initial weight 20.92 ± 0.56 g) fed for 8-week with diets having suboptimum (0.6) and optimal (2.3) DHA/EPA ratios. The analyses identified 358 up-regulated and 432 down-regulated differentially expressed genes, and 48 up-regulated and 30 down-regulated differential lipid molecules with 53% phosphatidylcholine, 14% phosphatidylethanolamine and 13% phosphatidylinositol. GO analysis, KEGG pathway enrichment and network of interaction analysis identified pathways of glycerophospholipid metabolism, PPAR signaling, oxidative phosphorylation, ribosome and thermogenesis as being significantly affected by dietary DHA/EPA ratio. Specifically, dietary DHA/EPA ratio regulated the expression of genes in lipid metabolism and PPAR signaling pathways such as fas, fabp-1/3, fatp, cptI and aco. Compared to the suboptimum ratio, the optimum dietary DHA/EPA ratio down regulated the gene experssion of phospholipase D1/2 and glycerol-3-phosphate O-acyltransferase in glycerophospholipid metabolism pathway, akt and s6 in mTOR pathway, ask1 in thermogenesis and non-alcoholic fatty liver disease pathway, and up regulated the pepck experssion in PPAR signaling pathway. Thus, dietary DHA/EPA ratio influence the lipid, protien and carbohydrate metabolism, immune response, and even the cell member structure. The findings improved our understanding of the impacts and molecular mechanisms of a nutritional challenge, in this case the dietary DHA/EPA ratio, increased basic knowledge of multi-omics analysis of in a commercially important crustacian species, the mud crab S. paramamosain , and provided the framework for further in-depth functional studies to promote sustainable aquaculture. • The study combined transcriptome and lipidome analysis in hepatopancreas of mud crab. • Optimal dietary DHA/EPA ratio led to reduced proinflammatory factors in mud crab. • The lipid metabolism in mud crab was regulated via PPAR signaling pathway. • DHA/EPA ratio may regulate cell membrane structure via glycerophospholipid metabolism. [ABSTRACT FROM AUTHOR]

Abstract: Over past decades, the farming of mud crab Scylla paramamosain has expanded rapidly in China. In our previous experiments, the results showed that the mud crab S. paramamosain in China could be divided into northern and southern populations and these two populations had differences in the adaptive capacity to cultivation environmental conditions. In order to verify this, the present study measured and compared the mitochondrial function and metabolic enzyme activity of these two populations during seasonal acclimatization. It was shown that the mitochondrial respiration rates in the gill, hepatopancreas and muscle of the northern population were significantly higher than those of the southern population in winter, spring and summer although the differences in summer were less pronounced than in winter and spring. In winter and spring, both state 2 and 3 mitochondrial respiration rates measured using either succinate or pyruvate+malate as the substrate in the gill of the northern population were consistently significantly higher than those of the southern population. The measurement of metabolic enzyme activity during different seasons showed that for the mitochondrial cytochrome C oxidase in the gill, hepatopancreas and muscle, the differences in activity between the northern and southern populations were in general consistent with those of the mitochondrial function. For example, the mitochondrial cytochrome C oxidase activities in the gill of the northern population were significantly higher than those of the southern population in winter, spring and summer. However, activity of lactate dehydrogenase, pyruvate kinase and hexokinase in the muscle were slightly different between the northern and southern populations. Only in summer the lactate dehydrogenase and pyruvate kinase activities in the muscle of the southern population were significantly higher than those of the northern population. In summary, the results of the present study showed that compared to the southern population, the northern population had a better compensation mechanism in mitochondrial metabolite level during cooler seasons, which might indicate that the northern population adapts better to low temperature than the southern population. Therefore, the temperature for the farming of the southern population and northern population should be different. [Copyright &y& Elsevier]

Pubertal molt is critical for the reproductive process of mud crab Scylla paramamosain , during which female crabs molt and mate with males, and subsequently, ovarian development is initiated. Since fasting occurs during pubertal molt, the mechanism underlying how the internal energy reserves support crabs' survival is unclear. In this study, the hepatopancreas and ovaries were collected for metabolomics analyses. The results show 11 carbohydrate and 4 lipid metabolic pathways were remarkably modulated in hepatopancreas, suggesting carbohydrates and lipids may be the main energy reserves in hepatopancreas postmolt. Further analysis showed that the levels of glucose, glucose 6-phosphate, glucose 1-phosphate, and fructose 6-phosphate were increased, indicating glycolysis was upregulated for energy supply. Meanwhile, 65 lipids were significantly changed, including 16 glycerophospholipids and 33 fatty acids, suggesting lipids might be another main energy supplier in the hepatopancreas. In the ovary, 7 carbohydrate and 1 lipid metabolic pathways were obviously affected, and 12 carbohydrates and 23 lipids were also significantly changed postmolt. Differently from the changes in the hepatopancreas, the glucose level was significantly decreased in the ovary, whereas the levels of eight glycerophospholipids and four unsaturated fatty acids were increased in the ovary but decreased in the hepatopancreas, suggesting lipids might be major energy reserves for ovarian development, and they were possibly exported from hepatopancreas. Moreover, the obvious changes in seven fatty acyl carnitines in the ovary indicate they were consumed via β-oxidation postmolt. Taken together, carbohydrates and fatty acyl carnitines were clearly the major energy reserves in pubertal molt. The hepatopancreas played a central role in the supply of energy reserves and nutrients. Numerous glycerophospholipids and unsaturated fatty acids may have accumulated in the ovary for the biosynthesis of egg yolk during ovarian development. This study suggests that proper increases in carbohydrate and lipid levels in crab feed ahead of pubertal molt may alleviate the effects of fasting and enhance successful reproduction. • Metabolites were notably modulated in both hepatopancreas and ovary postmolt. • Carbohydrates were major energy reserves in hepatopancreas during pubertal molt. • Fatty acyl carnitines were major energy reserves in ovary during pubertal molt. • Glycerophospholipids and unsaturated fatty acids were gathered in ovary postmolt. [ABSTRACT FROM AUTHOR]

This study investigated the influences of dietary zinc (Zn) on lipid and fatty acid metabolism of Scylla paramamosain. Three diets containing analyzed Zn levels including 44.5 (low Zn), 68.5 (medium Zn) and 254.7 (high Zn) mg/kg were prepared and fed to mud crab for 10 weeks, respectively. Medium dietary Zn levels improved the growth of mud crab than those fed the high Zn diet. The concentration of Zn in tissues was positively correlated with dietary Zn levels. Crabs fed with high Zn diet significantly decreased lipid content in hepatopancreas and muscle when compared to those fed the medium Zn diet. The lowest diameter, area and number of R cell in hepatopancreas histology were observed in those fed the high Zn diet. Furthermore, medium levels of dietary Zn significantly increased docosahexaenoic acid (DHA) and n-3 polyunsaturated fatty acid (PUFA) contents in hepatopancreas. Crabs fed high Zn diet dramatically down-regulated expression of acc , fas , g6pd , 6pgd and srebp , but up-regulated expression of aco3 , cpt1 and fabp1 in hepatopancreas. Crabs fed with medium Zn diet significantly increased the mRNA levels of fad in hepatopancreas. In summary, the results of present study emphasized the adverse effects of excess dietary Zn (254.7 mg/kg) on suppress hepatopancreatic lipid and n-3 PUFA accumulation, dietary 68.5 mg/kg Zn level was recommended to maintain optimal lipid and fatty acid metabolism of mud crab. • High levels dietary Zn caused lipid depletion in hepatopancreas and muscle of mud crab. • High levels dietary Zn restrained lipogenesis and promoted lipolysis of hepatopancreas. • Medium levels dietary Zn increased hepatopancreatic DHA and n-3 PUFA accumulation. [ABSTRACT FROM AUTHOR]

An 8 weeks regression study was performed to test the effects of increasing light intensities from darkness to 30 W m−2 on growth performance, molting, antioxidant capacity, and gene expression of molting and apoptosis-related genes in Scylla paramamosain. No significant differences were found in survival rates between treatments (ranging from 71.7 to 87.3% at the end of the experiment). However, weight gain and specific growth rate over the study period displayed a curvilinear response to light treatments with peak values in crabs exposed to 10 W m−2. Linear (BLM), quadratic (BQM), and 4-parameter saturation kinetic (4-SKM) models show the optimal light intensities for SGR were 12.98, 18.27, and 11.36 W m−2, respectively. The light intensity did not appear to impact molting. However, crabs reared in darkness showed significantly reduced molt frequency (3.51 ± 0.16) and extended intermolt intervals compared to other treatments. Melatonin levels in the eyestalks were significantly higher in crabs exposed to darkness (502.52 ± 56.24 pg mL−1) than light intensities of 10–30 W m−2 (413.50 ± 32.38 and 384.99 ± 15.56 pg mL−1). Cortisol levels were significantly lower in the 0 and 5 W m−2 groups. Light intensity significantly impacted the activity of antioxidant enzymes, with crabs showing a significant increase in total antioxidant capacity (T-AOC) under 10 W m−2, catalase (CAT), and superoxide dismutase (SOD) under 15 W m−2 and lower malondialdehyde (MDA). Gene expression of the molt-inhibiting hormone (MIH) was downregulated in eyestalks from crabs exposed to 10 W m−2 compared to darkness and 20–30 W m−2. Expression of apoptosis-related genes did not show clear light intensity trends. Taken together, these results suggest light intensity can impact S. paramamosain growth, molting, stress levels, and antioxidant capacity. As such, light regimes used in crab farming should be carefully considered to optimize productivity and welfare. • Light intensity ranging from 0 to 30 W m−2 had a significant effect on growth performance and molting of S. paramamosain. • The effects are mediated through hormonal, antioxidant and apoptosis pathways. • Optimal light intensity for growth of S. paramamosain is between 11.36 and 18.27 W m−2 at the water surface. [ABSTRACT FROM AUTHOR]

This study investigated the effects of different dietary egg yolk lecithin levels (with 0% (EL0), 1% (EL1), 2% (EL2), 3% (EL3), and 4% (EL4) EL supplementation) on survival, growth, lipid metabolism, and antioxidant capacity of the early juvenile green mud crab (Scylla paramamosain). Total of 420 crabs (initial weight 8.14 ± 0.30 mg) were randomly assigned into five experimental groups (with 3 replicates). At the end of the eight-week culture experiment, the highest survival (92.86 ± 4.52%) was obtained in EL3 group. Significantly higher growth performance (final body weight (FBW), weight gain (WG), and specific growth rate (SGR)) was also observed in the EL3 group, and there was no significant difference from the EL2 group. The highest molting frequency (MF) and transcription level of ecdysone receptor (EcR) gene were also obtained in EL2 group suggested that EL could increase the MF of early juvenile S. paramamosain by up-regulating the mRNA expression of EcR and ultimately promote its growth. In addition, the higher body lipid composition along with increased EL levels suggested that EL could promote lipid absorption and disposition in S. paramamosain. Compared with other crabs, the crabs in EL2 group had significantly higher ∑n-3/∑n-6 ratio. The highest transcription level of fatty acid binding proteins (FABPs) gene also observed in EL2 group. Furthermore, the crabs in EL4 group had significantly lower transcription level of fatty acid synthase (FAS) gene. This result implied that feeding of EL supress the fatty acids synthesis but induce fatty acid transport genes in S. paramamosain. In the hepatopancreas, the content of thiobarbituric acid reactive substances (TBARS) decreased significantly with the dietary EL addition. Meanwhile, significantly higher superoxide dismutase (SOD) activity was observed in the EL2 and EL3 groups. This indicated that dietary EL improved the antioxidant capacity of S. paramamosain. According to the polynomial regression analysis of SGR, 2.56% dietary EL level could achieve the maximum growth of early juvenile S. paramamosain. • 2.56% dietary egg yolk lecithin could achieve the maximum growth of S. paramamosain. • Dietary egg yolk lecithin suppress the FAS but induce FABPs genes in S. paramamosain. • Dietary egg yolk lecithin improved the antioxidant capacity of S. paramamosain. • Dietary egg yolk lecithin improved the nutritional value of S. paramamosain. [ABSTRACT FROM AUTHOR]