Functional Study on the BMP Signaling Pathway in the Molting of Scylla paramamosain

: 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 µ m/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.


Introduction
The mud crab (Scylla paramamosain), valued for its high market demand, serves as a crucial aquaculture species along the East Asian coastline [1].The molting process, which is essential for its growth and development, not only leads to physical enlargement but also signifies a transition in its physiological state [2,3].This intricate process involves a complex interplay of endocrine regulation and signal transduction mechanisms.Extensive research has elucidated the molting mechanism in crustaceans, highlighting the fundamental antagonistic interaction between molting hormone (MH) and molt-inhibiting hormone (MIH) [4].Furthermore, the regulation of calcium ions (Ca 2+ ) plays a critical role in this process [5].Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, are growth factors intricately involved in animal growth and development [6].In aquaculture, BMPs have demonstrated significant roles in biocalcification, tissue regeneration, and reproductive regulation.Studies have indicated that BMP2 and BMP4 show elevated expression levels in actively mineralizing tissues such as fish vertebrae and fins, underscoring their importance in biomineralization and skeletal development [7,8].Within bivalves, genes such as BMP2, BMP3, BMP7, BMP7b, and BMP10 are key players in shell damage repair and calcification [9,10].In Eriocheir sinensis, the expression of BMP2 and BMPR2 is significantly higher in the postmolt period than in the intermolt period [11].Furthermore, BMP2 and BMP7 are closely associated with germ cell generation and sperm maturation in both Eriocheir sinensis and S. paramamosain [12,13].Some studies have investigated the function of the BMP signaling pathway.In S. paramamosain, BMP2 has been shown to be involved in ovarian development and hormonal regulation [14], and Smad1 has been shown to be involved in immune response translation [15].However, there has been no research reported on the functional study of BMPs in the molting process of S. paramamosain.
The transduction of the BMP signaling pathway relies on the interaction between BMPs and their specific type I and type II receptors.This interaction initiates the activation of the intracellular Smad signaling pathway as well as diverse non-Smad pathways [16].Previous research has demonstrated that the inhibitor Dorsomorphin and its derivative LDN-193189 2HCl dose-dependently impede the phosphorylation of Smad1/5/8, thereby obstructing BMP signaling pathway transduction.However, Dorsomorphin also inhibits AMP-activated kinase (AMPK) and the receptor tyrosine kinases involved in PDGF and VEGF interactions [17].Our previous work initially screened the inhibitor LDN-193189 2HCl to determine the minimum concentration necessary to inhibit the BMP signaling pathway in S. paramamosain larvae.Subsequently, the larvae underwent a 30-day immersion culture at this concentration to explore the role of the BMP signaling pathway in mud crab molting, laying the groundwork for further investigations into the biological functions of this pathway in crustaceans.

Experimental Design and Management
The experimental protocol was approved by the Committee for Animal Conservation and Utilization at Ningbo University and conducted at the College of Marine Science at Ningbo University from September to October 2023.The megalopae of S. paramamosain were sourced from DaSheng Technology Co., Ltd.(Ningbo, China).
The experimental design for the inhibitor concentration screening was as follows: 2000 healthy, active, and intact megalopae were randomly distributed into 20 acrylic tanks (20 × 10 × 20 cm, 4 L).LDN-193189 2HCl was diluted in seawater to create five concentration groups: 0, 0.1 µmol/L, 0.5 µmol/L, 1 µmol/L, 2 µmol/L, and 4 µmol/L, with each treatment having four replicates.The larvae were fed frozen adult Artemia twice a day at 8:00 AM and 5:00 PM, with a complete water change every 48 h along with the addition of the inhibitor.The sampling time points were set at 6 h, 24 h, 48 h, 72 h, and 96 h.Samples were rinsed with PBS 2-3 times before being immediately frozen in liquid nitrogen for subsequent gene expression analysis.Survival rates and molting rates were calculated once all larvae in the control group had molted into juvenile crabs.Based on the above analysis, a suitable inhibitory concentration will be selected for subsequent experiments.
Subsequent daily management remained the same, and the rearing continued until all juvenile crabs in the experimental group had molted into the C IV stage.Samples were collected during the megalopa and C I stages at the time points of 6 h, 24 h, 48 h, and 72 h, during the premolt stage (24 h before molting) and the postmolt stage (C I soft-shelled crab).The weights were measured using an analytical balance 24 h after each molt.The full carapace width (FCW) and length (FCL) of the crabs were measured using an HDCE-X5 digital camera and ScopeImage 9.0 under a dissecting microscope (NiKang, Shanghai, China), while the average molting time and survival rates for each stage were recorded.Additionally, to investigate the role of the BMP signaling pathway in molting, the hepatopancreas tissues were collected during the intermolt stage of C III (C), the premolt stage of C III (D), and the postmolt stage of C IV (AB) and preserved in a freezer at −80 • C.

Gene Expression Analysis
Total RNA extraction was performed using a commercial RNA extraction kit (Omega Biotek, Norcross, GA, USA).Following extraction, 4 µL of the isolated RNA was utilized for agarose gel electrophoresis to verify the quality of the extraction.The electrophoresis conditions were set to 120 V and 140 mA for 10 min.Additionally, the RNA concentration and quality were assessed using a spectrophotometer (MaestroGEN, Taiwan, China).Upon confirming the high quality of the extracted RNA, the HiFiScript gDNA Removal RT MasterMix kit (Cwbio, Beijing, China) was used to eliminate the genomic DNA and synthesize cDNA. The

Calculations
The survival rate, molting rate, weight gain rate, and specific growth rate were calculated using the following formulas: survival rate(%) = N t /N 0 × 100; molting rate (%) = M t /N 0 × 100; where N t is the number of surviving larvae at the end of cultivation; N 0 is the initial number of larvae; M t is the number of C I juvenile crabs at the end of cultivation; W t is the average weight of crabs at the end of cultivation (g); W 0 is the average weight of megalopae at the start of cultivation (g); t is the duration of the experiment (d); WGR is the specific weight gain rate; and SGR is the specific growth rate.

Statistical Analysis
All data were analyzed using SPSS 22.0 and plotted using GraphPad Prism 9.0.The data were checked for normal distribution and variance homogeneity using the Kolmogorov-Smirnov test and Levene's test.One-way ANOVA (one-way analysis of variance) and independent samples t-tests were employed to compare the differences between treatment groups and control groups.Statistical significance was set at p < 0.05 for significant differences and p < 0.01 for highly significant differences.The growth parameters were expressed as the mean ± standard deviation (mean ± SD).

Survival and Molting Rate at Different Concentrations
The inhibitor at a concentration of 0.1 µmol/L showed no significant impact on the growth and molting of megalopae (p > 0.05).However, concentrations of 1 µmol/L and 2 µmol/L of the inhibitor affected the survival of the megalopae (Figure 1A,B), leading to a significant decrease in their survival rate.Furthermore, the inhibitor at a concentration of 2 µmol/L also resulted in a significant reduction in the molting rate of the megalopae (p < 0.05).At a concentration of 4 µmol/L, the inhibitor significantly reduced the survival and molting rates of the megalopae (p < 0.05).Under the influence of the inhibitor at 4 µmol/L, the megalopae could hardly survive and experienced a high mortality rate, with only a few individuals successfully molting.
molting rate (%) = Mt/N0 × 100; where Nt is the number of surviving larvae at the end of cultivation; N0 is the initial number of larvae; Mt is the number of C I juvenile crabs at the end of cultivation; Wt is the average weight of crabs at the end of cultivation (g); W0 is the average weight of megalopae at the start of cultivation (g); t is the duration of the experiment (d); WGR is the specific weight gain rate; and SGR is the specific growth rate.

Statistical Analysis
All data were analyzed using SPSS 22.0 and plotted using GraphPad Prism 9.0.The data were checked for normal distribution and variance homogeneity using the Kolmogorov-Smirnov test and Levene's test.One-way ANOVA (one-way analysis of variance) and independent samples t-tests were employed to compare the differences between treatment groups and control groups.Statistical significance was set at p < 0.05 for significant differences and p < 0.01 for highly significant differences.The growth parameters were expressed as the mean ± standard deviation (mean ± SD).

Survival and Molting Rate at Different Concentrations
The inhibitor at a concentration of 0.1 µmol/L showed no significant impact on the growth and molting of megalopae (p > 0.05).However, concentrations of 1 µmol/L and 2 µmol/L of the inhibitor affected the survival of the megalopae (Figure 1A,B), leading to a significant decrease in their survival rate.Furthermore, the inhibitor at a concentration of 2 µmol/L also resulted in a significant reduction in the molting rate of the megalopae (p < 0.05).At a concentration of 4 µmol/L, the inhibitor significantly reduced the survival and molting rates of the megalopae (p < 0.05).Under the influence of the inhibitor at 4 µmol/L, the megalopae could hardly survive and experienced a high mortality rate, with only a few individuals successfully molting.

Expression of ACVR1 at Different Concentrations
The inhibitor at a concentration of 0.1 µmol/L had no significant impact on the expression of the ACVR1 (p > 0.05) (Figure 2).However, the inhibitors at concentrations of 1 µmol/L and 2 µmol/L both significantly suppressed the expression of the ACVR1 (p < 0.05).Additionally, compared to the 1 µmol/L concentration group, the group treated with a concentration of 2 µmol/L exhibited an earlier onset and a more pronounced reduction in the expression of ACVR1.To ensure the effective inhibition of the BMP signaling pathway by the inhibitor LDN-193189 2HCL, a concentration of 2 µmol/L was selected for further research.0.05).Additionally, compared to the 1 µmol/L concentration group, the group treated with a concentration of 2 µmol/L exhibited an earlier onset and a more pronounced reduction in the expression of ACVR1.To ensure the effective inhibition of the BMP signaling pathway by the inhibitor LDN-193189 2HCL, a concentration of 2 µmol/L was selected for further research.

Expression of BMP-Related Genes at a Concentration of 2 μmol/L
The relative expression level of ACVR1 in the S. paramamosain megalopae treated with the 2 µmol/L inhibitor was significantly decreased at 24 h and 48 h (Figure 3A).Additionally, the expression levels of BMPRIB and Smad1 were significantly reduced at 48 h and 72 h compared with the control group.(p < 0.05) (Figure 3B,C).But after 72 h, this inhibitory effect weakened.The findings collectively suggest that treatment with 2 µmol/L LDN-193189 2HCL effectively inhibits BMP signaling pathway transduction in the megalopae of S. paramamosain.

Expression of BMP-Related Genes at a Concentration of 2 µmol/L
The relative expression level of ACVR1 in the S. paramamosain megalopae treated with the 2 µmol/L inhibitor was significantly decreased at 24 h and 48 h (Figure 3A).Additionally, the expression levels of BMPRIB and Smad1 were significantly reduced at 48 h and 72 h compared with the control group.(p < 0.05) (Figure 3B,C).But after 72 h, this inhibitory effect weakened.The findings collectively suggest that treatment with 2 µmol/L LDN-193189 2HCL effectively inhibits BMP signaling pathway transduction in the megalopae of S. paramamosain.

Growth Performance
The average weights of the megalopae in the control and experimental groups were 0.0049 ± 0.0010 g and 0.0048 ± 0.001 g, respectively.After a 30-day rearing period, the weight of the control group's juvenile crabs was significantly higher than that of the

Growth Performance
The average weights of the megalopae in the control and experimental groups were 0.0049 ± 0.0010 g and 0.0048 ± 0.001 g, respectively.After a 30-day rearing period, the weight of the control group's juvenile crabs was significantly higher than that of the experimental group starting from C II (Figure 4).The weight gain rate and average growth rate for the control group were 1859.67%± 82.43 and 9.91% ± 0.16, while for the experimental group, they were 736.73% ± 39.75 and 6.07% ± 0.11 (Table 2).Starting from the C II stage, the weight, full carapace length (FCL), full carapace width (FCW), weight gain rate (WGR), and specific growth rate (SGR) of the experimental group crabs were significantly lower than those of the control group (p < 0.01) (Tables 2 and 3).From the megalopa to the C IV stage, at each stage, the average molting interval of the control group was significantly lower than that of the experimental group (p < 0.05) (Figure 5).

Differential Gene Expression of the BMP Signaling Pathway across Molting Stages
To screen the key genes involved in molting in the BMP signaling pathway, the expression levels of the BMP genes during different molting stages were detected.The expression levels of ACVR1, BMP7, and BMPRIB remained consistently stable across the entire molting cycle (Figure 6D-F).In contrast, BMPR2, BMP2, and Smad1 displayed a distinct pattern of expression during the molting cycle, characterized by lower levels during the intermolt (C) and premolt (D) stages (Figure 6A-C).As molting advanced, there was a marked increase in their expression levels during the postmolt (AB) stage (p < 0.05).To screen the key genes involved in molting in the BMP signaling pathway, the expression levels of the BMP genes during different molting stages were detected.The expression levels of ACVR1, BMP7, and BMPRIB remained consistently stable across the entire molting cycle (Figure 6D-F).In contrast, BMPR2, BMP2, and Smad1 displayed a distinct pattern of expression during the molting cycle, characterized by lower levels during the intermolt (C) and premolt (D) stages (Figure 6A-C).As molting advanced, there was a marked increase in their expression levels during the postmolt (AB) stage (p < 0.05).

Expression of BMPR2, Smad1, and BMP2 at Different Molt Stages
LDN-193189 2HCL at a concentration of 2 µmol/L impacted the expression of BMPR2, Smad1, and BMP2 throughout the molting cycle.BMPR2 and Smad1 demonstrated significant downregulation during the intermolt stage compared to the control group (p < 0.05) (Figure 7A,C), with no significant variance in the premolt and postmolt stages (p > 0.05).BMP2 experienced significant downregulation in the postmolt stage in comparison to the control group (p < 0.01) (Figure 7B).(Figure 7A,C), with no significant variance in the premolt and postmolt stages (p > 0.05).BMP2 experienced significant downregulation in the postmolt stage in comparison to the control group (p < 0.01) (Figure 7B).

Molt-Related Gene Expression
The treatment with the 2 µmol/L inhibitor significantly increased the exp levels of MIH at 72 h, 96 h (p < 0.01), and during premolting (p < 0.05) (Figu Additionally, it significantly reduced the expression levels of EcR in both the prem stage (p < 0.01) and postmolting stage (p < 0.05) (Figure 8B).

Molt-Related Gene Expression
The treatment with the 2 µmol/L inhibitor significantly increased the expression levels of MIH at 72 h, 96 h (p < 0.01), and during premolting (p < 0.05) (Figure 8A), Additionally, it significantly reduced the expression levels of EcR in both the premolting stage (p < 0.01) and postmolting stage (p < 0.05) (Figure 8B).

Discussion
LDN-193189 2HCl has been extensively used to investigate the role of the BMP signaling pathway in tumorigenesis, stem cell differentiation, and biological development.For example, the BMP signaling pathway was suppressed by administering LDN-193189 to mice, and the involvement of the BMP signaling pathway in cancer dissemination was evaluated through the metastatic growth rate of cancer cells in bone [18].Furthermore, injections of LDN-193189 were administered to zebrafish embryos to investigate the role of the BMP signaling pathway in vascular development [17].
In this study, as the concentration of the inhibitor increased, there was a significant decrease in the molting and survival rates of the megalopae (Figure 1), along with a notable reduction in the expression level of ACVR1 (Figure 2).Previous studies have indicated that with an increase in the LDN-193189 2HCL concentration, there is a marked decrease in the vitality of Echinococcus multilocularis [19], accompanied by deformities and mortality, aligning with the findings of our study.Furthermore, treatment with the inhibitor at a concentration of 2 µmol/L resulted in a significant decrease in the expression levels of BMPRIB, ACVR1, and Smad1 in the megalopa larvae (Figure 3).The transduction of the BMP signaling pathway relies on the phosphorylation of Smad1/5/8 [20].While the activation of this phosphorylation process is dependent on the binding of the type I receptor BMPRIB, ACVR1 binds to the type II receptor [21,22].The result suggests that this concentration of inhibitor can effectively inhibit the BMP signaling pathway in S. paramamosain.

Discussion
LDN-193189 2HCl has been extensively used to investigate the role of the BMP signaling pathway in tumorigenesis, stem cell differentiation, and biological development.For example, the BMP signaling pathway was suppressed by administering LDN-193189 to mice, and the involvement of the BMP signaling pathway in cancer dissemination was evaluated through the metastatic growth rate of cancer cells in bone [18].Furthermore, injections of LDN-193189 were administered to zebrafish embryos to investigate the role of the BMP signaling pathway in vascular development [17].
In this study, as the concentration of the inhibitor increased, there was a significant decrease in the molting and survival rates of the megalopae (Figure 1), along with a notable reduction in the expression level of ACVR1 (Figure 2).Previous studies have indicated that with an increase in the LDN-193189 2HCL concentration, there is a marked decrease in the vitality of Echinococcus multilocularis [19], accompanied by deformities and mortality, aligning with the findings of our study.Furthermore, treatment with the inhibitor at a concentration of 2 µmol/L resulted in a significant decrease in the expression levels of BMPRIB, ACVR1, and Smad1 in the megalopa larvae (Figure 3).The transduction of the BMP signaling pathway relies on the phosphorylation of Smad1/5/8 [20].While the activation of this phosphorylation process is dependent on the binding of the type I receptor BMPRIB, ACVR1 binds to the type II receptor [21,22].The result suggests that this concentration of inhibitor can effectively inhibit the BMP signaling pathway in S. paramamosain.
In the hepatopancreatic tissues of S. paramamosain juvenile crabs at different molt stages, the expression of ACVR1, BMP7, and BMPR1B did not change significantly throughout the molt cycle, whereas BMP2, BMPR2, and Smad1 were significantly upregulated from the molt interphase.The results of the transcriptomic studies on the molt cycle of E. sinensis also showed that the expression of BMPR2 and ACVR1 was significantly upregulated after molting compared to the premolt stage [11].BMP2, BMPR2, and Smad1 may play a more important role in molting.
Most members of the BMP family have been shown to play important regulatory roles in biological calcification and tissue reconstruction [23].BMP2 is considered to be the sole factor capable of inducing bone formation independently and has been extensively studied in the formation of skeletons and cartilage in various animals [24].In mollusks, BMP2 has been shown to be associated with biomineralization and shell development in species such as Meretrix meretrix [25] and Pinctada fucata [26].As one of the receptors in the BMP signaling pathway, BMPR2 can bind to BMP2, 4, 6, and 7, and it has been reported that the physiological effects of BMPR2 depend on its combination with ligands [27].Mice with silenced Smad1 can still survive and reproduce; however, the combined loss of Smad1, 5, and 8 leads to severe cartilage developmental defects in mice.This suggests that Smad1 plays a primary role in signal transduction during cartilage formation and bone differentiation [28].In this study, BMPR2 and Smad1 expression in the hepatopancreas of juvenile crabs treated with the inhibitors was downregulated during the intermolt period, and BMP2 expression was significantly downregulated during the postmolt period.(Figure 7).During the molting process of crustaceans, the hepatopancreas serves as the primary storage site for calcium ions.From the premolt to postmolt stages, the calcium ion content the hepatopancreas reaches its peak and gradually decreases as the shell hardens [29].These results further suggest that the inhibition of the BMP signaling pathway may regulate molting by inhibiting calcium-ion-regulated processes in S. paramamosain.
The primary function of MIH is to regulate the molting process of crustaceans by inhibiting the synthesis of molting hormones in the Y-organ [30].Ecdysone receptor (EcR), the target of ecdysone, is present at the onset of the cascade of life processes in crustaceans such as molting, reproduction, and metamorphosis, and its expression varies significantly between molting stages [31].Existing studies have shown that the expression of MIH is significantly negatively correlated with body weight at different molt cycles in E. sinensis, while the expression of EcR is significantly positively correlated with body weight at different molt cycles [32].In addition, MIH has been shown to directly affect crustacean body weights [33].MIH is also important for maintaining the length of the intermolt period in crustaceans: the injection of a molt-inhibiting hormone significantly prolongs the molt time in Homaru americanus [34].In Metopograpsus messor, the expression of EcR fluctuated with its growing season and was significantly higher during the growing season, in which its weight gain rate was higher [35].In this study, treatment with 2 µmol/L LDN-193189 2HCL resulted in a reduction in the size and weight of the larvae crabs, along with a significant extension of the molting duration.Furthermore, it led to a notable increase in the expression of MIH (Figure 8A) and a significant decrease in the expression levels of EcR (Figure 8B).This suggests that the inhibition of the BMP signaling pathway may affect the expression of MIH and EcR, which leads to the inhibition of the growth process.

Conclusions
In summary, inhibiting the BMP signaling pathway with LDN-193189 resulted in the suppression of the molting process in the S. paramamosain.The key outcomes included a prolonged molting duration and reduced body weight and size.The BMP signaling pathway may regulate the molting physiological process through a combined effect on the expression levels of MIH and EcR and the process of exoskeleton hardening.Institutional Review Board Statement: This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Animal Ethics Committee of Ningbo University.In China, academic research on S. paramamosain is highly encouraged and does not necessitate particular permits.
Informed Consent Statement: Not applicable.

Figure 1 .
Figure 1.Survival rate (A) and molting rate (B) of S. paramamosain megalopae treated with different concentrations of inhibitors.Different letters indicate significant differences (p < 0.05).

Figure 2 .
Figure 2. Relative expression of ACVR1 in different concentrations of the inhibitor at 6 h, 24 h, 48 h, and 72 h.Different letters indicate significant differences (p < 0.05).

Figure 2 .
Figure 2. Relative expression of ACVR1 in different concentrations of the inhibitor at 6 h, 24 h, 48 h, and 72 h.Different letters indicate significant differences (p < 0.05).

Fishes 2024, 9 ,Figure 4 .
Figure 4. Average weight of experimental and control S. paramamosain.M, C I, C II, C III and C IV represent crablets at stages megalopa, I, II, III and IV.** represents a highly significant difference (p < 0.01).

Figure 4 .
Figure 4. Average weight of experimental and control S. paramamosain.M, C I, C II, C III and C IV represent crablets at stages megalopa, I, II, III and IV.** represents a highly significant difference (p < 0.01).

Figure 5 .
Figure 5. Average molting interval of S. paramamosain.M, C I, C II, and C III represent crablets at stages megalopa, I, II, and III.* represents a significant difference (p < 0.05).

Figure 5 .
Figure 5. Average molting interval of S. paramamosain.M, C I, C II, and C III represent crablets at stages megalopa, I, II, and III.* represents a significant difference (p < 0.05).

FishesFigure 8 .
Figure 8. Relative expression of (A) MIH genes and (B) ECR genes in experimental and control S. paramamosain megalopae at different time periods and molt stages.* represents a significant difference (p < 0.05).** represents a highly significant difference (p < 0.01).

Figure 8 .
Figure 8. Relative expression of (A) MIH genes and (B) ECR genes in experimental and control S. paramamosain megalopae at different time periods and molt stages.* represents a significant difference (p < 0.05).** represents a highly significant difference (p < 0.01).

Table 1 .
relative expression levels of ACVR1, BMPRIB, Smad1, MIH, EcR, BMP7, BMP2, and BMPR2 were analyzed using real-time quantitative PCR (LightCycler 480, Roche, Detroit, MI, USA) and the SYBR Premix Ex Taq kit (CWBIO, Beijing, China).The reaction mixture had a total volume of 20 µL (10 µL 2× Es Taq Master Mix, 8.2 µL ddH 2 O, 0.4 µL each primer, 1 µL cDNA).The primers used in this experiment are listed in Table1(Youkang Biological Technology Co., Ltd., Hangzhou, China).The real-time quantitative PCR conditions were as follows: pre-denaturation at 95 • C for 30 s; 40 cycles at 95 • C for 5 s and 60 • C for 30 s; and 95 • C for 15 s.The relative expression level of the target genes was calculated and analyzed using the equation 2 −∆∆CT .The specific primers used for real-time PCR in this study.

Table 2 .
The weight gain rate and specific growth rate of S. paramamosain.
W0: average weight of megalopae at the start of cultivation (g); Wt: average weight of crabs at the end of cultivation (g); WGR: specific weight gain rate; SGR: specific growth rate.** represents a highly significant difference (p < 0.01).

Table 3 .
Carapace length and carapace width of S. paramamosain juvenile crabs.

Table 2 .
The weight gain rate and specific growth rate of S. paramamosain.
W 0 : average weight of megalopae at the start of cultivation (g); W t : average weight of crabs at the end of cultivation (g); WGR: specific weight gain rate; SGR: specific growth rate.** represents a highly significant difference (p < 0.01).

Table 2 .
The weight gain rate and specific growth rate of S. paramamosain.
W0: average weight of megalopae at the start of cultivation (g); Wt: average weight of crabs at the end of cultivation (g); WGR: specific weight gain rate; SGR: specific growth rate.** represents a highly significant difference (p < 0.01).

Table 3 .
Carapace length and carapace width of S. paramamosain juvenile crabs.
C I, C II, C III and C IV represent crablets at stages I, II, III and IV; FCW: full carapace width; FCL: full carapace length; * represents a significant difference (p< 0.05), ** represents a highly significant difference (p < 0.01).

Table 3 .
Carapace length and carapace width of S. paramamosain juvenile crabs.