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Scylla paramamosain is an euryhaline species that is extensively distributed in coastal regions along the Indian and Pacific Oceans. Recently, S. paramamosain are considered as species that can be cultured in low-salinity areas to improve the local economy. However, the long-term effects and mechanisms of low salinity on its physiology remain unclear. In this study, S. paramamosain were reared in low-salinity (5 ppt) and normal sea-water environment (25 ppt) for 30 days. We compared haemolymph osmolality, gill structure, and transcriptome changes between the two groups. Results indicated that S. paramamosain subjected to a low-salinity environment showed gill damage such as broken lamellar and reduced haemocytes, some adaptive responses as increased mitochondria in chloride cells were also observed. Even with decreased water ion concentration in lower salinity, the concentrations of Na+, K+, and Cl- in the haemolymph of crab were remained higher than the surrounding water. Transcriptomic analysis revealed 2086 differentially expressed genes, including significant alterations in genes like hexosaminidase and neprilysin. Enriched pathways linked to ion transport were identified, highlighting the main adaptive mechanisms in long-term hypotonic environments. Our findings elucidated the effects and mechanism of long-term low salinity on gill structure and osmoregulation in S. paramamosain, providing theoretical basis for the development of low salinity aquaculture technology of this species.

To compare the allergenicity of native arginine kinase (nAK) and recombinant AK (rAK) from Scylla paramamosain and to identify the predominant allergenic domain of AK, AK was divided into 4 fragments: AK-E1 (amino acid (AA) 1–92), AK-E2 (AA 87–187), AK-E3 (AA 172–265), and AK-E4 (AA 276–357) based on the distribution of epitopes and the spatial structure of the AK molecule. The four recombinant fragments were expressed in the prokaryotic system Escherichia coli, and then nAK, rAK, and the recombinant fragments were purified. The allergenicity of recombinant proteins were evaluated using BALB/c mice. The results showed that the levels of specific antibodies in the serum and the secretion of Th2 type cytokines by the splenocytes of mice sensitized with rAK significantly increased, but the immunogenicity of rAK was weaker than that of nAK. Among the 4 fragments of AK, AK-E2 had the strongest immunogenicity. Meanwhile, rAK could stimulate RBL-2H3 cells to release β-hexokinase, but it was less effective than nAK. Among the 4 expressed fragments, AK-E2 and AK-E4 had a stronger stimulating effect on effector cells. In conclusion, the rAK expressed in the prokaryotic system showed weaker immunogenicity than nAK, and among the 4 fragments of AK, AA 87–187 has the strongest immunogenicity while AA 276–357 has the strongest immunoreactivity.

A 2 × 3 two-factor experiment was designed to assess the effects of dietary cholesterol (CHO) and phospholipids (PLs) on growth and lipid metabolism in early juvenile mud crabs (0.01 g crab−1). The experimental diets were designed with two CHO levels: 0.40 % (LCH) and 0.80 % (HCH), and three PLs levels: 1.80 % (LPL), 2.50 % (MPL), and 3.20 % (HPL). The 58-day aquaculture trial demonstrated that the LCH-HPL group achieved the best growth performance in mud crabs, characterized by the highest final body weight, weight gain, and specific growth rate. Regarding whole-body composition, dietary PLs increased total cholesterol (T-CHO), PLs, triglyceride (TG), and crude lipid content in the LCH groups. Mud crabs in the HCH groups had a higher proportion of saturated fatty acids, a lower proportion of monounsaturated fatty acids, and increased gene expression of sterol regulatory element binding protein 1c (srebp-1c) and fatty acid synthase (fas) compared to those in the LCH groups. As the dietary PLs increased, mud crabs in the LCH groups exhibited up-regulation in the expression of genes, including srebp-1c, fas, elongation of very long-chain fatty acid protein 4 (elovl4), elovl6, and Δ9-fatty acid desaturase (Δ9-fad). In the HCH groups, elevated dietary PLs resulted in the down-regulation of fatty acid binding protein (fabp) gene expression. In addition, high levels of dietary CHO and PLs (HCH-HPL) inhibited the catalase activity of mud crabs, resulting in a significant increase in the malondialdehyde content. Correlation analysis revealed that dietary CHO was positively correlated with the expression of long-chain polyunsaturated fatty acids synthesizing genes, and dietary PLs were positively correlated with the whole-body lipid content of mud crabs. Additionally, a positive correlation was found between the growth performance and whole-body content of crude protein, crude lipid, and antioxidant capacity in mud crabs.

The mud crab (Scylla paramamosain) is an important species for seawater aquaculture, the utilization of saline-alkali water provides potential for the further development of the mud crab industry, but the adaptation mechanism of the antennal gland to chloride type low-salt saline-alkali water has not been fully studied. In this study, we evaluated the osmoregulatory enzyme activity (n=3) and metabolic changes (n = 5) of the antennal gland in S. paramamosain after a sudden drop in salinity from 23 ‰ of seawater to 2 ‰ of chloride type low-salt saline-alkali water. After the decrease in salinity, the activity of osmoregulatory enzymes in the antennal gland, including Na+/K+-ATPase, NKCC, carbonic anhydrase (CA), and V-ATPase showed a trend of first increasing and then decreasing. Through LC-MS technology, we identified 308 differential metabolites. In addition, KEGG enrichment analysis identified eight significant metabolic pathways (P

Salinity affects the growth and development of crustaceans by affecting their physiological functions such as respiration, metabolism, osmotic regulation and immune defense. Gill is the most important organ of osmotic regulation. The adaptation of Scylla paramamosain to low salt is a process of oxygen consumption and energy consumption, and the mitochondria in the cells are considered to be the synthesis site of energy. Therefore, in this study, GC-MS technology was used to conduct non-targeted metabolomics studies on the posterior gill mitochondria of S. paramamosain under acute hyposaline stress, revealing the key role of mitochondria in gill response to acute low salinity stress. We identified 196 metabolites, 21 of which were differentially expressed. Differential metabolite KEGG enrichment analysis identified 16 metabolic pathways. The results of this study suggest that mitochondria may maintain osmotic homeostasis by increasing the utilization of monosaccharides, and decreasing the synthesis of amino acids in crabs under acute low salt stress when they are in a state of oxygen and energy depletion. In addition, carbohydrates and organic acid metabolites may also play an important role. For the first time, we analyzed the metabolic mechanism of posterior gill mitochondria in response to acute low salt, aiming to provide effective technical support and theoretical reference for the cultivation of S. paramamosain.

As rate-limiting enzymes of peroxisomal β-oxidation, acyl-coenzyme A oxidase (ACOXs) play vital roles in maintaining energy homeostasis and regulating reactive oxygen species (ROS) metabolism. However, there are no studies on the functions of ACOXs in crustaceans. In the present study, four full-length cDNA sequences of acoxs, namely the acox-1a (2403 bp), acox-1b (2733 bp), acox-3a (2878 bp) and acox-3b (3445 bp), were successfully isolated from mud crab Scylla paramamosain, which encoded 666, 673, 701 and 658 amino acids, respectively. Sequence analysis showed that the ACOX-1a, ACOX-1b and ACOX-3a possessed conserved structural domains like FAD-binding motif, fatty acyl CoA oxidase domain and peroxisomal targeting signal, while the ACOX-3b lacked peroxisomal targeting signal. Results of phylogenetic tree indicated that the four ACOXs of mud crab grouped gathered with their corresponding orthologues from crustaceans. The acox-1a, acox-3a and acox-3b were highly expressed in hepatopancreas, and the acox-1b was mainly distributed in muscle and hepatopancreas. Compared with feeding groups, the expression levels of acox-1a, acox-3a and acox-3b in hepatopancreas and acox-3a in muscle were markedly up-regulated in fasting groups, suggesting that the acoxs had significant effects in modulating energy balance during fasting. In addition, fasting significantly increased the transcriptional levels of nuclear factor erythroid 2-related factor (nrf2) and its downstream antioxidant genes (catalase (cat), glutathione peroxidase (gpx) and glutathione S-transferase (gst)) to improve antioxidant capacity for removing excessive ROS produced by ACOX-mediated peroxisomal β-oxidation. These results would be conducive to providing new insights into evolutionary characteristics and functions of acoxs in crustaceans.

Scylla paramamosain is an important mariculture animal. In the present study, we examined the physiological and biochemical characteristics of juvenile S. paramamosain under hypoosmotic stress and further verified the function of ABCC1, a key transport protein in gill tissue. The results showed that Na+/K+-ATPase, Ca2+-ATPase, and Na+/K+/2Cl- cotransporter in gill tissues increased first and then decreased. In addition, SOD and CAT activities also began to gradually increase, protecting gill tissues from oxidative damage. The hemolymph osmolarity decreased rapidly from 792 mOsm/kg to 560 mOsm/kg within 6 h, and then rose to 626.25 mOsm/kg at 48 h, and then remained stable. At the same time, Na+, Cl-, K+, and Ca2+ concentrations were decreased to varying degrees. There were significant changes in free amino acids such as aspartic acid, glutamic acid, and asparagine in hemolymph in response to acute salt depletion. By injection of ABCC1 inhibitor/activator, it was found that while ABCC1 transporter activity in the MK-571 group showed a decreasing trend at 9 h, Na+/K+ -ATPase activity began to increase and reached a peak at 48 h. Compared with the control group, the levels of aspartic acid, glutamic acid, asparagine, glycine, arginine, alanine, lysine, and proline in the MK-571 group showed significant differences at 6 h; Significant differences in glutamate, asparagine, arginine, alanine, lysine, and proline appeared at 12 h in the Thiethylperazine group. These results suggested that the dynamic changes of gill ion transport enzymes, antioxidant enzymes, inorganic ions, and free amino acids in hemolymph occurred in S. paramamosain under hypoosmotic stress, and the ABCC1 transporter played an important role in this regulatory process.

A six-week feeding trial was conducted to investigate the effects of dietary starch levels on growth performance, glucose and lipid metabolism and insulin signaling pathway of juvenile Scylla paramamosain. Three isonitrogenous (41.51% ± 0.31%) and isolipidic (8.68% ± 0.53%) experimental diets were formulated to contain 10%, 20% and 30% starch levels, respectively. A total 96 crabs were divided into 3 treatment groups. There were 4 replicates per treatment group and 8 crabs per replicate (initial weight 5.12 ± 0.05 g). The crabs fed the diet with 20% starch showed the best growth performance, with higher weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER), and feed efficiency (FE) compared to the other diets. Increasing starch levels in the diet resulted in higher levels of glycogen and lipid in the muscles and hepatopancreas. The levels of glucose (GLU), triglyceride (TG), and free fatty acid (FFA) displayed a significant increase in both hemolymph and hepatopancreas with the escalating dietary starch concentrations. In contrast, as the dietary starch levels increased, the concentrations of crustacean hyperglycemic hormone (CHH) and insulin-like peptides (ILPs) in hemolymph and hepatopancreas demonstrated a notable decline. The glycolysis and pentose phosphorylation pathways were significantly promoted, while the gluconeogenic pathway was significantly inhibited, with dietary starch levels increasing from 10% to 30%. Additionally, increasing dietary starch levels led to a significant down-regulation of genes related to the lipid synthesis, lipolysis and insulin signaling pathway. Histological results showed that hepatopancreas fat accumulation increased with the increase of dietary starch level. Overall, the findings of this study suggest that adding 20% starch to the diet can promote growth and improve nutrient utilization. In addition, the level of dietary starch had significant effects on the glucose and lipid metabolism and insulin signaling pathway of mud crabs, thus affecting the deposition of lipid reserves in hepatopancreas.

In mammals, fatty acid transport protein 1 (FATP1) plays important roles in cellular uptake and activation of long-chain fatty acid (LCFA), especially in processes of transportation, oxidation and triacylglycerol synthesis. However, the role of FATP1 in invertebrates, especially decapod crustaceans, is still poorly understood. In this study, the cDNA of a FATP1 gene from a decapod crustacean, mud crab Scylla paramamosain, was cloned and functionally characterized. The FATP1 gene encoded a polypeptide consisting of 643 amino acids that exhibits all the typical features of the FATP family and shares high homology with the other FATP orthologs of crustaceans. The relative mRNA expression levels of FATP1 were observed to be higher in metabolically active tissues such as hepatopancreas, stomach and gill than in other crab parts. Knockdown of the FATP1 mRNA in vivo significantly reduced triacylglycerols and total lipid levels in the hepatopancreas, accompanied by an increase in the expression of genes related to fatty acid transportation, allocation and hydrolysis, including long-chain acyl-CoA synthetase 3/4 (ACSL3/4) and carnitine palmitoyl transferase 1 (CPT1), and a decrease in the expression of genes related to fatty acid synthesis such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in the hepatopancreas. Furthermore, increased dietary n-3 long-chain polyunsaturated fatty acid (LC-PUFA) levels resulted in the up-regulation of the FATP1 expression in the hepatopancreas, accompanied by an increase in LC-PUFA content, especially eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), in both polar (PLs) and neutral lipids (NLs) in the hepatopancreas and muscles of crabs. These findings suggested that the FATP1 gene identified in S. paramamosain might play important roles in regulating long-chain fatty acid metabolism and deposition in crustaceans.

Lipid accumulation in ovaries is essential for reproductive success in decapods. Understanding of the mechanism of ovarian lipid accumulation is still in its infancy. In this study, we investigated lipid compositions of ovaries and hepatopancreas throughout vitellogenesis and hemolymph in the late-vitellogenic stage in the mud crab Scylla paramamosain. Total lipid content in ovaries consecutively increased from the early to late stage, and the main accumulated lipids were phosphatidylcholine (PC), alkenylglycerophosphoethanolamine (alkenyl-PE), triacylglycerol (TG), cholesteryl ester, and cholesterol. The lipid composition of hemolymph indicated that PC, alkenyl-PE, diacylglycerol (DG), ether DG, monoacylglycerol, and cholesterol were the main lipids transferred to ovaries from hepatopancreas. In hepatopancreas, the total lipid content appeared to increase during vitellogenesis and the late stage was significantly different from early and mid-stages. TG was the predominant lipid class that slightly decreased from the early to mid-stage, but dramatically increased in the late stage. In contrast, PC and alkenyl-PE significantly increased from the early to mid-stage and remained constantly in relatively low proportion until the late stage. These findings suggested PC and alkenyl-PE in the hepatopancreas likely play critical roles in regulating lipid transfer. Esterified fatty acids in ovaries and hepatopancreas were mainly C16:0, C18:0, 16:1, C18:1, C18:2, C20:4, C20:5, and C22:6, of which highly unsaturated fatty acids (HUFA) primarily occurred in alkenyl-PE and phosphatidylserine (PS) but not PC. In hemolymph, PC was abundant in HUFA besides of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA), while PS was predominantly composed of SFA and MUFA, implying a transformation of HUFA between phospholipids prior to transfer. These findings highlight the importance of phospholipid metabolism in the hepatopancreas to ovarian lipid accumulation. Our study provides novel insights into the mechanism of ovarian lipid accumulation in decapods.

The feeding rhythm is one of the key factors determining the success of artificial breeding of S. paramamosain. To understand the feeding rhythm of the different zoea larva developmental stages of S. paramamosain, the feeding rate, digestive enzyme activity, and expression of metabolism-related genes were investigated in the present study. The results showed that the S. paramamosain feeding rate has strong diurnal feeding rhythm, being significantly higher at 10:00–14:00 from stages ZI to ZIV. While the feeding rate peaked at 14:00 on Days 10 and 11, the peak shifted to 18:00 on Day 12. The activity of digestive enzymes amylase, pepsin and lipase decreased at night but increased in the daytime, showing a single-phase rhythm similar to that of the feeding rate, suggesting that the digestive enzyme activity was closely associated with the feeding rate during the larval development. Compared to pepsin and lipase, the activity of amylase was the most consistent with feeding rate. In particular, amylase activity peaked at 18:00 on Day 12. Due to its synchronicity with feeding activity, the activity of amylase could provide a potential reference for determining the best feeding time during zoea stages in S. paramamosain breeding. Moreover, the relative mRNA expression of metabolism-related genes SpCHH and SpFAS at most tested points was lower from 10:00 to 14:00, but higher at 18:00 to 6:00 of the next day. On the other hand, the expression patterns of SpHSL and SpTryp were converse to those of SpCHH and SpFAS. Our findings revealed that the S. paramamosain zoea has an obvious feeding rhythm, and the most suitable feeding time was 10:00–18:00 depending on different stages. The feeding rhythm is a critical aspect in aquaculture, influencing a series of physiological functions in aquatic animals. This study provides insights into the feeding rhythm during the zoea development of S. paramamosain, making a significant contribution to optimizing feeding strategy, improving aquafeed utilization, and reducing the impact of residual feed on water environment.

As rate-limiting enzymes of β-oxidation of fatty acids in mitochondria, the carnitine palmitoyltransferase (CPT) played an important role in regulating energy homeostasis of aquatic animals. However, there was very little research on β-oxidation of fatty acids in crustaceans. In the present study, the full-length cDNA sequences of cpt-1a, cpt-1b and cpt-2 were isolated from the hepatopancreas of Scylla paramamosain, and contained 4206, 5303 and 3486 bp respectively. Sequence analysis showed that the CPT-1A, CPT-1B and CPT-2 encoded proteins with 777, 775 and 672 amino acids respectively, and only the CPT-1A possessed a transmembrane region. In addition, both the CPT-1B and CPT-2 contained conservative functional domains like N-terminal domain and acyltransferases choActase 2, while the CPT-1A lacked. The results of phylogenetic tree indicated that the CPT-1A, CPT-1B and CPT-2 of S. paramamosain gathered together with their corresponding orthologues from crustaceans. The tissue distribution exhibited that the cpt-1a was highly expressed in hepatopancreas, followed by muscle, eyestalk and cranial ganglia, and the muscle, eyestalk and heart were main expressed tissues of cpt-1b. Furthermore, the high expression levels of cpt-2 were mainly detected in hepatopancreas, muscle and heart. The transcriptional levels of cpt-1a, cpt-1b and cpt-2 were significantly up-regulated under chronic low salinity stress. Besides, at the acute low salinity stress condition, the expression levels of cpt-1a, cpt-1b and cpt-2 in hepatopancreas were dramatically increased in 14‰ and 4‰ salinity groups at the 6h and 48h, while the transcriptional levels of cpt-1a, cpt-1b and cpt-2 in muscle were signally up-regulated in 14‰ and 4‰ salinity groups at the 12h and 24h, showing an alternate response pattern. Similarly, the present study found that fasting could markedly increase the expression levels of cpt-1a, cpt-1b and cpt-2 in hepatopancreas and muscle, especially cpt-1a in hepatopancreas as well as cpt-1a and cpt-1b in muscle. The results above indicated that the cpt-1a, cpt-1b and cpt-2 played a crucial part in providing energy for coping with fasting and salinity stress. These results would contribute to enhancing the knowledge of cpt phylogenetic evolution and their roles in energy metabolism of crustaceans.

The mud crab Scylla paramamosain is a key species in China due to its high nutritional value and great economic worth and has grown in popularity. Myo-inositol can modulate versatile physiological functions in aquatic animals. In the present study, S. paramamosain megalopa were given graded concentrations of myo-inositol (0, 1, 2, 4, and 8 ppm) by water immersion to explore how their metamorphosis would be affected. The results showed that supplementing with myo-inositol remarkably increased transformation and survival rate from megalopa to crablet by at least 1.16 and 1.26 times, respectively. To decipher the molecular mechanism of how myo-inositol increases metamorphosis and survival rate, we further performed transcriptome-based gene expression profiling of both megalopa and crablet treated with myo-inositol. The integrative transcriptome analyses predicted that the differentially expressed genes (DEGs) were significantly enriched in chitinase activity, structural constituent of cuticle, and chitin binding, which are associated with the decomposition and reconstruction of cuticle. qPCR results confirmed that myo-inositol mediated gene expression levels of the above cuticle-related pathways. Considering the importance of the cuticle in exoskeleton formation and molting, it can be concluded that myo-inositol-induced changes in the cuticle decomposition and reconstruction might have accelerated the transformation from megalopa to crablet of S. paramamosain. Besides, numerous DEGs were significantly enriched in protein digestion and absorption, amino sugar and nucleotide sugar metabolism. It implies that myo-inositol may improve survival by regulating energy or nutrient absorption. Additionally, the accelerated metamorphosis by myo-inositol may improve survival from megalopa to crablet of S. paramamosain. Overall, this study will provide the first insights into the underlying molecular mechanisms by which myo-inositol increases metamorphosis and survival.

In this study, we added LDN-193189 2HCL to inhibit the BMP signaling pathway in Scylla paramamosain and then explored the function of this pathway in molting through the changes in the growth performance and molt-related gene expression. The study findings indicated that the expression of ACVR1, BMPRIB, and Smad1 in Scylla paramamosain was suppressed when the LDN-193189 2HCL concentration in the culture water was 2 µm/L. Subsequently, following a 30-day experiment, there was a significant reduction in the molting frequency, growth rate, and body size of the S. paramamosain larvae. An analysis of the BMP pathway gene expression during the molting phase revealed that the BMP2, BMPR2, and Smad1 genes displayed cyclic expression patterns, while ACVR1, BMP7, and BMPRIB maintained consistent expression levels throughout the molting cycle. Additionally, the expression levels of BMP2, BMPR2, and Smad1 in the inhibition group were significantly lower compared to those in the control group. Furthermore, the inhibition of the BMP pathway led to an increase in the expression of MIH during the intermolt period and a decrease in the expression of EcR during the premolt period. These findings demonstrate that the BMP signaling pathway affects the molting of Scylla paramamosain juvenile crabs by influencing the expression of the critical genes MIH and ECR during molting, offering valuable data for functional research on the BMP signaling pathway in crustaceans.

Apoptosis Inhibitor 5 (API5) is a widely concerned nuclear protein with diverse functions in organisms, so far, study of API5 is still quite limited in lower animals, and its role in viral immune response has not been addressed. Here, we explored the function of API5 in mud crab (Scylla paramamosain) during White Spot Syndrome Virus (WSSV) infection. The interacting protein Hsp20 of API5 was screened by pull-down assay, and API5 and hsp20 were knocked down by RNAi interference. The results showed that API5 was upregulated along with virus infection, silencing of API5 led to increased WSSV copy numbers and apoptotic rate of hemocytes, highlighting its significance in the immune response. Moreover, we discovered a novel interaction between API5 and Heat Shock Protein 20 (Hsp20), and then revealed that Hsp20 could promote cell apoptosis of hemocytes and reduce viral copy numbers by suppressing API5. The current study therefore improves the knowledge of API5-Hsp20 axis and provides novel insights into intricate mechanisms governing the antiviral response in marine crustaceans.

To investigate the embryonic development of the mud crab Scylla paramamosain, we analyzed three critical parameters: egg color of, embryo morphology (through conventional and laser scanning confocal microscopy), and the distribution of cell divisions. During embryonic development, the egg color exhibited a progressive transition, shifting from orange to reddish-orange, then to brown, before ultimately darkening to black. Each embryo displayed a spherical shape, measuring approximately 280 μm in diameter, characterized by a smooth surface devoid of any depressions. The embryonic cell division was in the form of mixed oogenesis, comprised of complete division in the early stage, spiral oogenesis in the middle stage and surface division in the late stage. It is noteworthy that the blastopore appeared at the position where the transparent area and cell aggregation just appeared under the microscope, and the blastomere was a characteristic of the embryo entering the gastrulation stage. After entering the gastrulation stage, the cells aggregated towards the blastopore and formed two symmetrical cell clusters, which formed a V-shape with the void of the classic blastopore. When the transparent region occupied approximately 1/5 of the embryo’s volume, the embryo entered the nauplius stage, and the thoracic and abdominal armor, as well as the optic lobe and abdominal limb primordia, could be clearly distinguished. The appearance of the compound eye pigment band indicated the stage of compound eye pigment formation. At this time, the transparent area accounted for 1/4 of the embryo and a large number of ganglia appeared. The change of the compound eye pigment band from red to black was also one of the reasons for the blackening of the egg color of the crabs. The data obtained through this study have potential applications in the determination of embryonic development status and obtaining of high-quality seeds for S. paramamosain culture.

Dietary intake is an essential source of energy and nutrients, and plays an irreplaceable role in the breeding of S. paramamosain seedlings. In this study, live Artemia nauplii (LA), compound feeds (CF), frozen copepods (FC) and frozen adult Artemia (FA) were utilized as feed during the megalopa stage. To determine the impact that diet has on the metabolism of crablets that underwent metamorphosis from the megalopa stage, analyses of both metabolomics and fatty acid content were conducted. In the LC-MS-based metabolomics analysis, a total of 104, 205 and 83 significantly different metabolites (SDMs) were identified after being fed with FC, FA, and CF, respectively, as compared to the LA group. Furthermore, significant differences in KEGG compounds among the three comparisons exhibited similarity and were mainly associated with categories such as “Lipids”, “Hormones and transmitters”, and “Peptides”. The fatty acid content analysis indicated that the ΣMUFA was significantly higher in the LA and CF groups compared to the other two groups. In contrast, the highest level of ΣPUFA was found in the LA group. In addition, the CF group showed significantly higher expression levels of the fatty acid synthesis genes, FAS and ACC. Conversely, the expression level of the fatty acid decomposition-related gene CPT1 was the highest in the LA group. In comparison with the FA group, the expression level of FABP3 was significantly decreased in the LA and CF groups. In summary, there were significant differences observed in the metabolic profiles of crablets that metamorphosed from the megalopa under different diets. Our experimental results suggested that LA is more advantageous in the cultivation of the S. paramamosain megalopa compared to the other three diets. While it remains a diet that cannot be entirely substituted at present, LA has the potential to improve the culture performance of the S. paramamosain megalopa. The current study could provide valuable data into the development of artificial diets necessary for the future of the mud crab seedling breeding industry.

IntroductionThe mud crab, Scylla paramamosain, holds great commercial significance as a marine crustacean widely cultivated in the Indo-Pacific region. Understanding the core gut microbiota of aquatic animals is crucial for their overall health and growth, yet the core gut microbiota of mud crab remains poorly characterized.MethodsIn this study, we gathered gut samples from mud crabs across five locations within Sanmen Bay, China. Through the utilization of high-throughput sequencing, we delved into the composition of the gut microbial community and identified the core gut microbiome of mud crab.ResultsOur results demonstrate that the gut microbial diversity of mud crab did not exhibit significant variation among the five sampling sites, although there were some differences in community richness. At the phylum level, we identified 35 representative phyla, with Firmicutes, Proteobacteria, Bacteroidota, and Campilobacterota as the dominant phyla. Among the 815 representative genera, we discovered 19 core genera, which accounted for 65.45% of the total sequences. These core genera were distributed across 6 phyla, and among them, Photobacterium exhibited the highest average relative abundance.DiscussionPhotobacterium has probiotic activity and may play a crucial role in enhancing the immune response of the host and maintaining the diversity of the gut microbiota. Moreover, we observed a positive correlation between the relative abundance of core genera and the stability of the gut microbial community. Furthermore, our findings revealed distinct differences in gut microbial composition and specific taxa between the sexes of mud crab. These differences subsequently influenced the functionality of the gut microbial community. Overall, our investigation sheds light on the core gut microbiota of mud crab, emphasizing the importance of core gut microbial communities in maintaining the health and growth of these commercially significant marine crustaceans.

Providing shelters is considered an effective method to prevent self-harm and improve the survival rate of Scylla paramamosain. In this study, four shelter groups—the shelters of which included fine sand, arched tiles, PVC pipes, and trapezoidal net cages—and one group without shelters were established to investigate the effects of shelters on S. paramamosain by calculating their feeding rate, molting rate, survival rate, and growth index. The results showed that the feeding rates of the fine sand and PVC pipe groups were greater than 12% and that the feeding rates of the arched tile and trapezoidal net cage groups were greater than 9%, while the feeding rate of the group without shelters was only 6.7%. The food conversion rates of both the fine sand and PVC pipe groups exceeded 21%. In contrast, the food conversion rate of the non-shelter group was the lowest at 14%. The molting rates of the fine sand and PVC pipe groups were the highest, reaching 29% in the later stages of the experiment, while the molting rate of the non-shelter group was only 19%. There was no significant difference in survival rates among the groups during the first 20 days of the experiment. However, on the 60th day, the survival rate of the fine sand group was 92%, while the non-shelter group had a survival rate of 79%. The experimental results showed that the incidence of cheliped injuries in the fine sand group was 16%, while it was 25% in the non-shelter group. During the daytime, the occupancy rate of burrows by the four shelter groups was 60–70%, while during the night, the occupancy rate of each shelter decreased to 40–50%, and there was no significant difference in occupancy rates among the shelter groups during the night. The weight and full carapace width of S. paramamosain in the fine sand and PVC pipe groups were significantly higher than those in the other groups (P< 0.05), and the weight gain rate and specific growth rate of the fine sand and PVC pipe groups were significantly higher than those of the other groups (P< 0.05). Research has shown that fine sand and PVC pipes, as shelters for S. paramamosain, can effectively prevent cannibalism, increase feeding rates and survival rates, and promote growth and molting. This study provides scientific guidance for the proper selection of shelters in the farming process of S. paramamosain.

Introduction: Elongation of very long-chain fatty acids protein 6 (ELOVL6) played crucial roles in regulating energy expenditure and fatty acid metabolism. Many studies have performed to investigate the physiological roles and regulatory mechanisms of elovl6 in fish and animals, while few studies were reported in crustaceans.Methods: Here we reported on the molecular cloning, tissue distribution and expression profiles in response to dietary fatty acids, ambient salinity and starvation stress in Scylla paramamosain by using rapid amplification of cDNA ends (RACE) and quantitative real-time PCR.Results: Three elovl6 isoforms (named elovl6a, elovl6b and elovl6c) were isolated from S. paramamosain in the present study. The complete sequence of elovl6a was 1345 bp, the full-length sequence of elovl6b was 1419 bp, and the obtained elovl6c sequence was 1375 bp in full length. The elovl6a, elovl6b and elovl6c encoded 287, 329 and 301 amino acids respectively, and exhibited the typical structural features of ELOVL protein family members. Phylogenetic analysis showed that the ELOVL6a from S. paramamosain clustered most closely to ELOVL6 from Portunus trituberculatus and Eriocheir sinensis, while the ELOVL6b and ELOVL6c from S. paramamosain gathered alone into a single branch. Quantitative real-time PCR exhibited that the relatively abundant expression of elovl6b was observed in intestine and stomach, and the elovl6a and elovl6c were highly expressed in hepatopancreas. In addition, studies found that replacing fish oil with soybean oil could significantly increase the transcriptional levels of three elovl6 in hepatopancreas of S. paramamosain, and the expression of elovl6a and elovl6c in hepatopancreas were more sensitive to dietary fatty acids than the elovl6b. Compared with the normal sea water group (27‰), the expression of sterol-regulatory element binding protein1c (srebp-1), elovl6a, elovl6b and elovl6c were upregulated in the low salinity groups, particularly in 7‰. On the contrary, the starvation stress suppressed the expression of srebp-1, elovl6a, elovl6b and elovl6c.Discussion: These results may contribute to understand the functions of elovl6 in fatty acid synthesis and regulatory mechanisms in crustaceans.

Saline–alkali water represents a huge underdeveloped resource due to its harsh properties of high pH, high salinity, and complex ionic composition. In our previous research, the mud crab, Scylla paramamosain could adapt to the saline–alkali environment, which may bring dual benefits to high-quality aquaculture and the utilization of saline–alkali water. Yet, no systematic study related to S. paramamosain's ability to carbonate alkali, the key gene responsible for adaptation to the harsh environment, has been reported. In this study, we investigated the mud crab's response to a wide range of carbonate–alkali waters, verifying the specific gill tissue with the highest carbonic anhydrase (CA) expression. Afterward, we cloned the CA gene in S. paramamosain (Sp-CA) for the first time. The cDNA sequence is 1085 bp, with a 55-bp 5′-untranslated region (UTR) and 214-bp 3′-UTR. The open reading frame (ORF) of 816-bp nucleotides encodes a polypeptide of 271 amino acids (AAs). In silico analysis and phylogenetic analysis of crustacea's CAs were performed, elucidating the unique function and evolutionary relation of Sp-CA. We also conducted short-term and long-term stress experiments with mud crabs in a saline–alkali environment, with the relative mRNA expression and DNA methylation profile of Sp-CA under such stress having been clarified, bringing new insight into the understanding of mud crabs' adaptation and tolerance to a saline–alkali environment.

Variability in food availability leads to condition-dependent investments in reproduction. This study is aimed at understanding the metabolic response and regulatory mechanism of female Scylla paramamosain in response to starvation in a temporal- and tissue-specific manner. The mud crabs were starved for 7 (control), 14, 28, and 40 days for histological and biochemical analysis in the hepatopancreas, ovary, and serum, as well as for RNA sequencing on the hepatopancreas and ovary. We further highlighted candidate gene modules highly linked to physiological traits. Collectively, our observations suggested that starvation triggered endogenous ovarian maturation at the expense of hepatopancreas mass, with both metabolic adjustments to optimize energy and fatty acid supply from hepatopancreas to ovary in the early phase, followed by the activation of autophagy-related pathways in both organs over prolonged starvation. These specific adaptive responses might be considered efficient strategies to stimulate ovarian maturation of Scylla paramamosain under fasting stress, which improves the nutritional value of female mud crabs and other economically important crustaceans.

Anesthesia serves as an effective method to mitigate the stress response in aquatic animals during aquaculture and product transportation. In this study, we assessed the anesthetic efficacy of clove oil, tricaine methane-sulfonate (MS-222), ethanol, and magnesium chloride by anesthesia duration, recovery time, 24-hour survival rate, and the behavior of mud crabs (Scylla paramamosain). Additionally, the optimal anesthetic concentration for varying body weights of mud crabs was also investigated. The results revealed that clove oil emerged as the optimal anesthetic for mud crabs, with a 24-hour survival rate surpassing those observed in MS-222 and magnesium chloride treatments. Ethanol caused amputation and hyperactivity in mud crabs. Regression analyses between the optimal anesthetic concentration of clove oil and the weight categories of 0.03–27.50 g and 27.50–399.73 g for mud crabs yielded the following equations: y = 0.0036 x3 − 0.1629 x2 + 1.7314 x + 4.085 (R2 = 0.7115) and y = 0.0437 x + 2.9461 (R2 = 0.9549). Clove oil exhibited no significant impact on serum cortisol, glucose, lactate content, aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities, or superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels in mud crabs across different treatment groups. Anesthesia induced by clove oil in mud crabs resulted in an increase in inhibitory neurotransmitters such as glycine. However, the recovery from anesthesia was associated with elevated levels of the excitatory neurotransmitters L-aspartic acid and glutamate. In conclusion, clove oil proves to be a safe and optimal anesthetic agent for mud crabs, exerting no physiological stress on the species.

Three isonitrogenous diets (41% crude protein) with 4.87%, 10.75% and 16.10% lipid were designed to evaluate the effects of dietary lipid levels growth, antioxidant capacity, expression levels of genes related to lipid and glucose metabolism of juvenile Scylla paramamosain. Each diet was fed to four replicates of 32 crabs with initial weight 5.10 ± 0.05 g for 6 weeks. The results indicated that crabs fed diet with 10.75% lipid exhibited significantly higher percent weight gain (PWG) and specific growth rate (SGR) than those fed diet with 4.87% lipid (P

IntroductionThe intestinal microbiota acts as an additional “organ” that performs a variety of fu\nctions for the host’s health. However, the composition and role of the intestinal microbiota in Scylla paramamosain cultivated in inland low salinity saline-alkaline water are unknown.MethodsAccordingly, from the perspective of practical production, we explored the intestinal microbiota communities and the critical bacteria of S. paramamosain in normal salinity seawater (NS), coastal low salinity seawater (CS), acute low salinity seawater (AS) and inland low salinity saline-alkaline water (IS).ResultsResults showed that there were significant differences in the diversity composition of intestinal microbiota and the relative abundance of dominant taxa in each group of cultured crabs. Firmicutes, Proteobacteria, Bacteroidota and Campilobacterota were shown to be the major phyla shared by the four groups, with Bacteroidota having the highest relative abundance (27.10%) in the inland low salinity saline-alkaline water group (IS). Fusobacteriota had the highest proportion in IS group compared with other low salinity groups. A total of 284 indicator bacteria were identified, belonging to eight phyla, and their relative abundances were varied significantly (P < 0.05). Genus Carboxylicivirga, as the indicator bacterium of the IS group, may play a critical role in the adaptation of crab to saline-alkaline water environment. Moreover, salinity may exert considerable selective pressure on the entire microbial community.DiscussionThese findings revealed the features of the intestinal microbiome in S. paramamosain in multiple low salinity patterns, and provided candidate probiotics and basic information for crab farming in saline-alkaline water, which was conducive to the development and perfection of mud crab culturing technology in inland low salinity saline-alkaline water.

Hexavalent chromium (Cr6+) is a metallic contaminant that in organisms and poses a great threat to biological health and ecology due to its toxicity and biomagnification. However, the potential health risks to mud crabs and humans from dietary excessive Cr6+ were currently unknown. Therefore, a 9-week feeding trial was conducted to evaluate the toxic effects of dietary Cr6+ levels (1.4, 77.5 and 312.2 mg/kg) for mud crab (Scylla paramamosain). The results indicated that dietary Cr6+ levels had no significant effect on growth performance for mud crab. Cr concentrations in hepatopancreas and muscle significantly increased with dietary Cr6+ levels increasing from 1.4 to 312.2 mg/kg. The antioxidant capacity of mud crab was enhanced by increased the activities of superoxide dismutase (SOD) and scavenging capability for hydroxyl free radical (SCHR) to resist the Cr6+ toxicity. However, there was no statistical difference in the rate of apoptosis in hemocytes among all treatments. Expression levels of caspase 2 gene related to apoptosis in hemocytes significantly up-regulated with dietary Cr6+ level increasing from 1.4 to 312.2 mg/kg. For food safety, crabs fed diets with 77.5 and 312.2 mg/kg Cr6+ had target hazard quotient (THQ) values greater than 1 for the hepatopancreas, indicating a serious risk for human consumption. According to the target cancer risk (TCR) analysis, people who consumed muscle in mud crab fed diet with 312.2 mg/kg Cr6+ were at unacceptable cancer risk. Overall, the results of the present study indicated mud crab had a strong resistance to Cr6+ toxicity, and dietary excessive Cr6+ did not decline growth performance and survival, oxidative stress and apoptosis in mud crab. Excess Cr6+ were accumulated in tissues such as hepatopancreas and muscle, and consumption of crabs tissues fed diets with 77.5 and 312.2 mg/kg Cr6+ will provoke health and cancer risk in humans.

Although human gC1qR is a multi-ligand binding protein with diverse biological functions, the functions of invertebrate gC1qR homologues remain largely unknown. In the present study, we characterized a novel gC1qR homologue, namely SpgC1qR, from mud crab Scylla paramamosain. SpgC1qR shared high identity and similar three-dimensional structure with human gC1qR. After challenge with White spot syndrome virus (WSSV), the transcripts of SpgC1qR were significantly increased, suggesting that SpgC1qR may be involved in antiviral immune response. To reveal the likely antiviral activity of SpgC1qR, the proliferation profile of WSSV in SpgC1qR-silenced crabs was examined. The result showed that knockdown of SpgC1qR by RNAi facilitated viral proliferation in vivo. This result was further confirmed by a SpgC1qR pre-incubation assay, in which pre-incubating WSSV particles with rSpgC1qR dramatically suppressed viral replication. Moreover, a GST pull-down assay revealed that SpgC1qR specifically bound to the viral envelope protein VP28. These findings clearly demonstrated that SpgC1qR specifically interacted with viral envelope protein VP28 and restricted WSSV replication, suggesting that it played a crucial role in anti-WSSV immune response of mud crab. This study provided new insights into the antiviral mechanism mediated by SpgC1qR and the biological functions of invertebrate gC1qR homologues.

This study investigated the effects of different dietary arginine (Arg) levels on the growth, protein synthesis, antioxidant capacity, and immunity of postlarval mud crab Scylla Paramamosain. Six isonitrogenous and isolipidic diets were formulated to contain 1.51%, 1.81%, 2.16%, 2.35%, 2.73%, and 3.07% dietary Arg levels (dry matter). There were four replicates for each diet treatment (26 crabs per replicate, initial body weight: 7.40 ± 0.15 mg). After eight weeks of feeding trial, the survival and molting frequency (MF) of crabs were not affected by the experimental treatment (P>0.05). Crabs fed the 2.50% Arg diet achieved the highest weight gain (WG) and specific growth rate (SGR) (P

Molting is a key biological process in crustaceans, with impacts on their growth, development and reproduction. Extracellular vesicles (EVs) serve as bio-cargo carrying such as nucleic acids, proteins, and lipids which mediate intercellular communication and participate in various cell biological processes. In this study, we obtained hemocyte transcriptome data during the intermolt, premolt, and postmolt stages of the mud crab Scylla paramamosain. We analyzed the differentially expressed genes in the three stages of molt and identified a number of immune-related genes and structural cuticle genes. We then isolated and characterized the EVs from the hemolymph of S. paramamosain and sequenced their miRNA. In total, we characterized 89 EVs microRNAs (miRNAs) targeting 1447 genes that are potentially involved in the molting process of S. paramamosain. The EVs miRNAs mainly regulated the immune-related genes and affected the molting process by mediating ecdysone signaling and insulin signaling pathways during the mud crab molt cycle. This is the first study of EVs miRNAs and their interaction with mRNA to identify important candidate genes associated with, or regulating, the molting process. This study provides a better understanding of, and novel insights into, the molting cycle of crustaceans and offers baseline information for further studies in the mechanisms regulating molt.

Antimicrobial peptides (AMPs) may be the most promising substitute for antibiotics due to their effective bactericidal activity and multiple antimicrobial modes against pathogenic bacteria. In this study, a new functional gene named Spgillcin was identified in Scylla paramamosain, which encoded 216 amino acids of mature peptide. In vivo, Spgillcin was dominantly expressed in the gills of male and female crabs, offering the highest expression level among all tested organs or tissues. The expression pattern of Spgillcin was significantly altered when challenged by Staphylococcus aureus, indicating a positive immune response. In vitro, a functional truncated peptide Spgillcin177–189 derived from the amino acid sequence of Spgillcin was synthesized and showed a broad-spectrum and potent antibacterial activity against several bacterial strains, including the clinical isolates of multidrug-resistant (MDR) strains, with a range of minimum inhibitory concentrations from 1.5 to 48 μM. Spgillcin177–189 also showed rapid bactericidal kinetics for S. aureus and Pseudomonas aeruginosa but did not display any cytotoxicity to mammalian cells and maintained its antimicrobial activity in different conditions. Mechanistic studies indicated that Spgillcin177–189 was mainly involved in the disruption of cell membrane integrity where the membrane components lipoteichoic acid and lipopolysaccharide could significantly inhibit the antimicrobial activity in a dose-dependent manner. In addition, Spgillcin177–189 could change the membrane permeability and cause the accumulation of intracellular reactive oxygen species. No resistance was generated to Spgillcin177–189 when the clinical isolates of methicillin-resistant S. aureus and MDR P. aeruginosa were treated with Spgillcin177–189 and then subjected to a long term of continuous culturing for 50 days. In addition, Spgillcin177–189 exerted a strong anti-biofilm activity by inhibiting biofilm formation and was also effective at killing extracellular S. aureus in the cultural supernatant of RAW 264.7 cells. Taken together, Spgillcin177–189 has strong potential as a substitute for antibiotics in future aquaculture and medical applications.

Reactive oxygen species (ROS), a group of unstable and highly reactive molecules or free radicals, are productions of molecular oxygen through cell metabolism. In invertebrates, especially in crustaceans, hemolymph plays an extremely important role in innate immune regulation and inflammatory responses, in which various microbes were modified by ROS. Even so, the information about ROS in relation to hemolymph microbiota and innate immunity is still limited in mud crab. To investigate the underlying mechanism, the ROS-excessive mud crabs (Scylla paramamosain) caused by knocking down catalase (SpCAT) were used to demonstrate the effects of abnormal levels of ROS on the changes in the hemolymph microbiota. Our results showed that SpCAT silence significantly increase ROS production and decrease the load of the bacteria in the hemolymph in mud crab. In the siCAT group, the dominant phyla were Proteobacteria, Actinobacteria, Acidonacteria, Bacteroidetes, Firmicutes, Gemmatimonadetes, Chloroflexi, Planctomycetes, Rokubacteria and Verrucommicrobia. Among them, the relative abundance of Firmicutes and Cyanobacteria was significantly increased upon the elevated ROS production. Some bacterial genera such as Bacteroides, Parabacteroides, Faecalibacterium and Romboutsia also increased with higher level of ROS. Moreover, the results showed that excessive ROS could promote the evident autophagy of the hemocytes. The result of this study demonstrated that ROS plays a vital role in modulating the homeostasis of the microbiota, and SpCAT is also crucially important in the ROS-mediated hemolymph microbiota homeostasis and the autophagy of the hemocytes in mud crab.

An eight-week feeding trial was conducted to evaluate the influence of dietary arachidonic acid (20:4n-6) on growth performance, fatty acid composition and antioxidant capacity on postlarva mud crab (Scylla paramamosain). Five isonitrogenous and isoenergetic diets with 0.34% (the control diet), 0.48%, 0.84%, 1.18% and 1.81% ARA levels were formulated by adding ARA rich oil in the basal diet. There were triplicate groups of 28 postlarval crabs (initial weight 8.15 mg) for each diet treatment. The results indicated that crabs fed diets with 1.18% ARA showed significantly higher final body weight (FBW), weight gain (WG) as well as specific growth rate (SGR) than the other groups. Survival, molting frequency (MF) and intermoult period were not significantly affected by different dietary ARA addition. Compared with the stable DHA content in crab, the EPA content decreased significantly with increase of dietary ARA addition. In crabs, superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities generally increased with dietary ARA supplementation, and were highest in 1.18% dietary ARA group. The MDA (malondialdehyde) concentrations showed the opposite trend. In addition, with the increase of dietary ARA content, the relatively increased lipid content in body composition was observed. The gene expression of fatty acid synthase (fas) and fatty acid binding protein-3 (fabp-3) in crabs increased with increasing ARA level in diets (0.34–1.18%) and then down-regulated expression levels with further ARA addition (1.81%). These results suggested that moderate dietary ARA level (1.18%) contributed to the improvement of growth performance and antioxidant capacity, and regulated the fatty acid composition and gene expression of some lipid metabolism in crabs, while excessive ARA addition in diets may cause oxidative stress of postlarval Scylla paramamosain. Moreover, the dietary ARA/EPA ratio of 1.82 may be suitable for the growth of postlarval Scylla paramamosain.

Scylla paramamosain is an important marine aquaculture crustacean on the southeastern coast of China. Given the difficulties of overcrowded coastal aquaculture space and insufficient production, inland saline-alkaline water culture has the potential to alleviate this bottleneck. This study separated 600 crabs into four groups: normal salinity (12-18 ppt, NS), coastal low salinity (3-5 ppt, CS), inland low salinity saline-alkaline (1.5 ppt, IS), and acute low salinity (23 ppt down to 3 ppt, AS), followed by a transcriptomic analysis of the gills. CS-vs-NS, IS-vs-NS, and AS-vs-NS obtained 1154, 1012, and 707 DEGs, respectively. GO analysis showed that the DEGs of the three comparison groups were mainly involved in cellular process, metabolic process, biological regulation, organelle, membrane, extracellular region, binding, and catalytic activity. The findings demonstrate that a significant number of genes are engaged in controlling metabolic processes in the osmoregulation process, and that cell membrane catalysis and different enzymes play a vital part in the environmental adaption process. KEGG enrichment analysis revealed that IS possesses a considerable number of signaling pathways that play important roles in osmoregulation when compared to NS. The cAMP signaling pathway increased the expression of CaM and Na+/K+-ATPase. These findings show that cations like Ca2+, Na+, and K+ are critical for S. paramamosain to adapt to inland low salinity saline-alkaline water, and that the cAMP signaling pathway regulates their levels. This research provides a theoretical foundation for improving the saline-alkaline aquaculture technology of S. paramamosain.

Scylla paramamosain is a high-quality cultivar for saline-alkaline water aquaculture as a euryhaline crustacean species. However, salinity impacts the respiratory metabolism, growth, and survival of marine crustaceans. The metabolic response of crabs adapting to multiple low salinity environments has not been thoroughly studied yet, especially in inland saline-alkaline water. In this study, we analyzed metabolites in the gill and hemolymph of crabs cultured in three different low salinity environments. The results showed that membrane composition (lipids and lipid molecules) and free amino acids played an essential role in the osmoregulation of crabs, and the energy consumption accompanied as well. Meanwhile, S. paramamosain relied on ion transport and energy metabolism under acute/short-term low salinity conditions for osmoregulation. In contrast, amino acids and energy metabolism occupied a leading position in long-term low salinity. Furthermore, taurine and hypotaurine play a vital role in crabs adapting to inland saline-alkaline water. This is the first study to identify the crucial metabolites and key pathways as biomarkers to differentiate the metabolic mechanisms of S. paramamosain under multiple low salinity stress modes based on GC-MS technology, which provided novel insight into the metabolic response of S. paramamosain adapting to inland low salinity saline-alkaline water, and provided theoretical guidance for the aquaculture of S. paramamosain in the inland saline-alkaline water.

C-type lectins are pattern recognition receptors (PRRs) involved in innate immunity. They possess one or more carbohydrate recognition domains (CRDs) belonging to Ca2+-dependent carbohydrate binding proteins. Herein, two C-type lectins were identified from the mud crab Scylla paramamosain. The open reading frame (ORF) of SpCTL-E encodes 160 amino acids including a CRD and a signal peptide, while the SpCTL-F ORF encodes a 244 amino acid polypeptide with a signal peptide and a CRD, and the key motifs for carbohydrate binding specificity are Asp-Pro-Asn (DPN) and Gln-Pro-Asp (QPD), respectively. DPN is a newly discovered motif, while QPD is typical of C-type lectins. The binding specificity toward different pathogen-associated molecular patterns (PAMPs) was analysed, and both bound three PAMPs: lipopolysaccharide (LPS), glucan (GLU) and peptidoglycan (PGN). SpCTL-F bound more strongly than SpCTL-E. SpCTL-E and SpCTL-F bound to five and seven microbial species, respectively, both agglutinated Gram-negative bacteria and fungi, and both promoted the phagocytosis activity of hemocytes toward Escherichia coli, and both enhanced the encapsulation activity of hemocytes towards Ni-NTA beads. SpCTL-E inhibited the growth of Micrococcus luteus and E. coli, while SpCTL-F also inhibited Vibrio parahaemolyticus. Thus, SpCTL-E and SpCTL-F are important PRRs with a broad nonself-recognition spectrum and play a key role in pathogen elimination.

This study investigated the combined effects of feeding ration and cheliped autotomy on the intermolt duration, molting success, molt increments in size and weight, and ecdysteroid receptor gene (SpEcR) expression of early juvenile mud crab Scylla paramamosain. Newly molted second stage juvenile crabs (C2) were subjected to four feeding conditions; optimal, suboptimal (1/2 optimal), low (1/4 optimal) ration and starvation, and autotomy (intact vs. cheliped autotomy) in a 4 × 2 factorial design until all crabs successfully molted or died. A significant interaction of feeding ration and cheliped autotomy on intermolt duration was identified. With reduced feeding ration, both intact and cheliped autotomized crabs showed increased time and desynchrony of molting, but decreased carapace size and body weight. Importantly, all crabs with different feeding rations even the low ration had high rates of molting success (> 95%), while the crabs subjected to starvation died without molting. When fed optimal ration, the mean intermolt duration of the cheliped autotomized crabs was significantly prolonged, while no such effect was found between autotomized and intact crabs subjected to suboptimal or low feeding ration. The qRT-PCR revealed that the expression of SpEcR showed a general trend of inhibited by reduced feeding ration, which was consistent with observed significantly increased intermolt duration. Interestingly, the transcript level of SpEcR was only significantly affected by cheliped autotomy under the optimal and suboptimal feeding rations but not for the low feeding ration. Together, the results of this study suggest that the S. paramamosain early juveniles have a strong tolerance for fluctuations in food availability. In addition, the availability of food and limb autotomy could significantly affect growth, molting duration and synchrony of the crabs, which appeared to reflect in SpEcR expression level that involved in the regulation of molting and limb regeneration process of the juvenile crabs.

The mud crab, Scylla paramamosain, has abundant nutrients in its edible parts, ovary, hepatopancreas, and muscle during the ovarian maturation stage. The ovary of S. paramamosain can re-mature after spawning during the secondary ovarian maturation period. We aimed to analyze the characteristics of the first vitellogenesis period (FVP)1 and second vitellogenesis period (SVP)2 of S. paramamosain during ovarian maturation to understand the differences in vitellogenesis patterns between the first and second ovarian maturation periods. Accordingly, the gonadosomatic index (GSI) and hepatopancreatic index (HSI), the external and histological characteristics of the ovary and hepatopancreas, the Sp-Vg (vitellogenin, Vg) expression levels in the hepatopancreas and ovary, and the dynamics of the biochemical components in the ovary, hepatopancreas, and muscle were determined. Based on the results, the GSI was significantly positively correlated with HSI during the FVP and significantly negatively correlated with HSI from stage Ⅳ to stage Ⅴ of the SVP. A significant difference was found between the FVP and SVP in the hepatopancreas. Notably, the hepatopancreas displayed a gradual degeneration trend during the SVP. The expression level of Sp-Vg was significantly higher in the hepatopancreas than that in the ovary during the FVP and SVP. Seventeen amino acids were detected in the hepatopancreas, ovary, and muscle during the FVP and SVP, with glutamate as the predominant amino acid. During the FVP and SVP, the C16:0 and C18:1n9c were the dominant fatty acids in the hepatopancreas and ovary, the MUFA gradually increased in the ovary and hepatopancreas, and a significant difference was found in the dynamic trend of the HUFA and SFA contents from stage Ⅳ to stage Ⅴ between the FVP and SVP. These findings indicate that the ovary can re-mature after spawning in S. paramamosain and can maintain the status of the first ovarian maturation; however, the hepatopancreas gradually degenerate during the SVP.

Ubiquitination is an essential post-translational modification of proteins and involved in many physiological processes, including sperm differentiation, oocyte maturation, and embryonic development. Among them, the ubiquitin-conjugating enzyme E2 plays a crucial role in gametogenesis. However, the participation of E2 in the regulation of the gonad development of crustaceans is still very limited, especially for oogenesis. In this research, we isolated and identified full-length complementary DNA of ubiquitin-conjugating enzyme E2 from mud crab (Scylla paramamosain) (Sp-Ubc2), detected distribution in different tissues by real-time quantitative PCR, and described expression patterns during oogenesis by in situ hybridization. The results demonstrated that the deduced proteins with a ubiquitin-conjugating (UBC) catalytic domain was clustered with other arthropods. In addition, the Sp-Ubc2 was widely distributed in different tissues of female individuals and kept the highest expression level in the ovary. Moreover, the expression level of Sp-Ubc2 changed significantly during oogenesis and reached peak in secondary and tertiary vitellogenesis stages. At the same time, the Sp-Ubc2 signal in the ovary gradually concentrated in nuclei. These results indicated that Sp-Ubc2 might play important roles in the cell cycle of oogenesis. This study would provide insights into the regulatory mechanism of gonad development in S. paramamosain, and it also enriched the theoretical basis of ubiquitin family genes involved in crustacean gonad development.

Ecdysis triggering hormone (ETH) plays an important role in molting, reproduction, and courtship behavior in insects. To investigate the potential downstream pathways and genes of ETH in Scylla paramamosain, RNA interference (RNAi) was conducted on crabs at early (D0) and late (D2) premolt substages, and the transcriptome profiles of each group were compared by RNA sequencing. Real-time quantitative polymerase chain reaction (RT-qPCR) and semiquantitative polymerase chain reaction (RT-PCR) results showed a significant knockdown of ETH at D0 stage, whereas a significant increase was shown conversely in crabs at D2 substage after the injection of dsETH. A total of 242,979 transcripts were assembled, and 44,012 unigenes were identified. Transcriptomic comparison between crabs at D2 and D0 substages showed 2,683 differentially expressed genes (DEGs); these genes were enriched in ribosome and pathways related to transcription factor complex and cell part. Twenty DEGs were identified between dsETH-injected and dsGFP-injected crabs at D0 substage; these DEGs were involved in carbohydrate metabolism, one carbon pool by folate, and chitin binding. Twenty-six DEGs were identified between dsETH-injected and dsGFP-injected crabs at D2 substage; these DEGs were involved in calcium channel inhibitor activity, fat digestion and absorption, and cardiac muscle contraction. RT-qPCR verified the differential expression of the selected genes. In conclusion, crabs at D0 substage are more active in preparing the macromolecular complex that is needed for molting. Moreover, ETH has potential roles in carbohydrate metabolism, one carbon pool by folate, and chitin binding for crabs at D0 substage, while the role of ETH turns to be involved in calcium channel inhibitor activity, fat digestion and absorption, and cardiac muscle contraction at D2 substage to facilitate the occurrence of molting. The selected DEGs provide valuable insight into the role of ETH in the regulation of crustacean molting.

Nghiên cứu nhắm đánh giá khả năng thay thế Artemia bằng thức ăn chế biến (TACB) để ương ấu trùng cua biển Scylla paramamosain từ Zoe 3 lên cua 1. Nghiên cứu được thực hiện với 2 thí nghiệm: ương từ giai đoạn Zoe 3 lên megalop và từ megalop lên cua 1. Mỗi thí nghiệm gồm 6 nghiệm thức, được bố trí ngẫu nhiên với 5 lần lặp lại. Thí nghiệm được bố trí với chế độ cho ăn 5 lần/ngày cho tất cả các nghiệm thức, nghiệm thức đối chứng cho ăn hoàn toàn Artemia, nghiệm thức TACB cho ăn hoàn toàn thức ăn chế biến. Bốn nghiệm thức còn lại được thay thế từ 1- 4 lần Artemia bằng thức ăn chế biến, Kết quả tỷ lệ sống của zoae 5 và megalopa có khuynh hướng tăng dần theo tỷ lệ cho ăn Artemia, trung bình lần lượt là 30,8-91,1% và 8,6-17,0% trong đó nghiệm thức 3, 4 và 5 lần Artemia/ngày khác biệt không ý nghĩa thống kê. Tỷ lệ sống từ megalopa đến cua 1 dao động 57,6-76,8%, trong đó nghiệm thức hoàn toàn TACB đạt cao. Khi sử dụng thức ăn chế biến ương cua lượng Artemia giai đoạn Zoea3 đến megalopa giảm được 40% và giai đoạn megalopa đến cua 1 có thể giảm đến 100% lượng Artemia.

Khảo sát được thực hiện 2 đợt: đợt 1 vào năm 2015 và đợt 2 vào năm 2020 bằng cách phỏng vấn trực tiếp nông hộ sản xuất giống cua biển (Scylla paramamosain) ở 3 tỉnh ĐBSCL có nghề sản xuất giống cua phát triển mạnh gồm Bạc Liêu, Cà Mau và Kiên Giang nhằm thu thập thông tin về kỹ thuật sản xuất giống cua biển, tình hình sử dụng thức ăn trong ương ấu trùng cua biển. Mật độ ương ấu trùng cua giai đoạn zoea1 trong khoảng 101,6 và 192,7. Tỷ lệ sống megalopa và cua 1: megalopae trung bình 8,17%, và cua 1 là 6,74%. Kết quả khảo sát cho thấy 100% các trại sản xuất giống cua sử dụng kết hợp ấu trùng Artemia với thức ăn nhân tạo cho tôm để ương ấu trùng cua biển. Hàm lượng protein thức ăn dao động từ 42-52% và lipid từ 7 đến 14,2%. Chi phí thức ăn Artemia chiếm tỷ lệ cao (75%) và chi phí thức ăn nhân tạo chỉ chiếm 3%. Sản xuất giống cua biển ở Đồng bằng sông Cửu Long còn phụ thuộc nhiều vào nguồn Artemia và chưa có thức ăn chuyên cho ương giống cua biển, vì vậy việc nghiên cứu sản xuất thức ăn ương ấu trùng cua biển là cần thiết cho việc phát triển nghề sản xuất giống cua biển.

Mesanophrys sp. is reported to be highly pathogenic to marine crustaceans. This study presents the first report of Mesanophrys sp. infection in the mud crab (Scylla paramamosain). In this study, we first recorded the survival rates of an experimentally infected group and a control group; the cumulative survival rate in the infected group was significantly lower compared to the control group after 72 h (73.20% vs. 94.19%), while the highest mortality of S. paramamosain occurred within the first 24 h post-infection. Then, we investigated the dynamic distribution and tissue tropism of the Mesanophrys sp. in the infected S. paramamosain by a quantitative real-time polymerase chain reaction (qPCR). The result showed that a significant increase in the number of Mesanophrys sp. could be detected in all tested tissues (obtained from the eyestalks, gills, heart, nerves, muscles and hepatopancreas) at 3 h post-infection. The numbers of Mesanophrys sp. in the gill, eyestalk and nerve tissues were relatively higher than in the other tissues. The gill tissue showed the highest numbers from 6 to 48 h. Histopathological observation found a severe collapse in the filament structure, which indicated tissue-specific pathogen infection. Furthermore, the antioxidant enzyme activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in three representative tissues (gill, muscle and hepatopancreas) were compared between the infected and control groups, and a significant increase in enzyme activity was observed in all three tested tissues in the infected group, indicating a relatively strong innate immune defense reaction that could have been induced by Mesanophrys sp. infection. These results will be helpful to Mesanophrys sp. pathogenicity-related research and the control of this pathogen in S. Paramamosain in the future.

In the study, a new gene homologous to the known antimicrobial peptide Scygonadin was identified in mud crab Scylla paramamosain and named SCY3. The full-length sequences of cDNA and genomic DNA were determined. Similar to Scygonadin, SCY3 was dominantly expressed in the ejaculatory ducts of male crab and the spermatheca of post-mating females at mating. The mRNA expression was significantly up-regulated after stimulation by Vibrio alginolyticus, but not by Staphylococcus aureus. The recombinant protein rSCY3 had a killing effect on Micrococcus luteus and could improve the survival rate of mud crabs infected with V. alginolyticus. Further analysis showed that rSCY3 interacted with rSCY1 or rSCY2 using Surface Plasmon Resonance (SPR, a technology for detecting interactions between biomolecules using biosensor chips) and Mammalian Two-Hybrid (M2H, a way of detecting interactions between proteins in vivo). Moreover, the rSCY3 could significantly improve the sperm acrosome reaction (AR) of S. paramamosain and the results demonstrated that the binding of rSCY3, rSCY4, and rSCY5 to progesterone was a potential factor affecting the sperm AR by SCYs on. This study lays the foundation for further investigation on the molecular mechanism of SCYs involved in both immunity and physiological effects of S. paramamosain.

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.

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.