Based on metabolomics analysis: metabolic mechanism of intestinal tract of 
Scylla paramamosain (mud crab) is widely distributed in China, Vietnam, Japan, Thailand, Philippines, Indonesia, and so on. It has a delicious taste and high nutritional value, and is one of the important economic crabs for aquaculture . In recent year, the total production of mud crab has been declining, and the breeding areas are faced with land shortage and shortage of breeding production, which needs to be solved urgently. S. paramamosain can survive and grow in a wide range of salinities , which is an excellent variety suitable for saline-alkali water aquaculture. As a species with high economic value and strong adaptability to the environment, its cultivation under low-salt conditions can not only improve the utilization efficiency of saline-alkali land, but also provide new possibilities for the sustainable development of aquaculture. At present, the mud crab has been successfully cultured in the inland saline-alkali water areas of Henan, Shanxi and Ningxia in China , which played an important role in expanding the breeding space of mud crab in China and improving the economic and social benefits of saline-alkali water utilization. Meanwhile, salt-alkali land mud crab farming has expanded the area of mud crab farming to some extent, increasing the output of mud crabs and alleviating the problem of insufficient output. In the context of global climate change and ecological environment deterioration, the traditional aquaculture industry is facing various challenges . Saline-alkali land, as an ecosystem with specific environmental pressures, shows unique potential for adaptive farming. Saline-alkali soil and water resources are abundant. There are more than 800 million hectares of saline-alkali land in the world . Currently, China has about 98 million hectares of saline-alkali land, which is about 1/8 of the world’s saline-alkali land area . This is particularly important for the development and utilization of saline-alkali land. These saline and alkaline soil and water resources can be divided into three types according to the difference in ion composition: sulfate, carbonate and chloride types . The saline-alkali land in coastal areas of China is mainly chloride saline land. The exploitation and utilization of saline-alkali soil and water will fully relieve the pressure of coastal aquaculture. In recent years, the cultivation of mud crab in saline-alkali soil has been carried out in many places in China , and the physiological studies on low salinity and alkali cultivation of mud crab have been published one after another. For example, Hu et al.  and Che et al.  discussed the effects of sulfates and bicarbonates on the mud crab, respectively, but the relevant analysis on the cultivation of mud crab in coastal chlorinated low salinity saline-alkali soil pond has rarely been reported. Therefore, through the metabolomics analysis of the intestinal tract of mud crab, the adaptation of mud crab to coastal chlorinated saline-alkali water was explored in this paper, which provided important reference and practical significance for saline-alkaline soil and water culture of mud crab. 

Data check Using non targeted metabolomics analysis, the metabolic changes in the intestinal tract of the S. paramamosain under long-term chloride salt alkali water stress were studied. LC/GC-MS detected a total of 4113 metabolites. To verify the validity of the data, OPLS-DA discriminant analysis statistical method (Fig. ) was used to improve the analytical ability and effectiveness of the model, maximizing the differences between different groups within the model. According to the OPLS-DA score plot, it can be seen that there is a significant separation between the control group (CK) and the long-term chloride type saline-alkali water stress group (LSAW group) (Fig. A and B), indicating that LSAW group leads to significant changes in the metabolite profile of the S. paramamosain . Through the fitting test of the OPLS-DA model, it can be found that the explanatory power (R 2 Y (cum)) and predictive power (Q 2 (cum)) of the model for the sample are 0.999 and 0.919 (GC-MS) and 0.994 and 0.906 (LC-MS), respectively (Fig. C and D). In addition, the slope of the straight line is relatively large, and the intercept of Q 2 is -0.243 (GC-MS) and − 0.183 (LC-MS), indicating that the OPLS-DA model does not exceed the fit (Fig. ). In summary, the OPLS-DA model has good explanatory and predictive abilities, and can reflect the differences between different sample groups in real responses. The above results indicate that compared with the CK group, there were significant changes in the metabolic products of the intestinal tract in the LSAW group of S. paramamosain . A is LC-MS OPLS-DA scores plots; B is GC-MS OPLS-DA scores plots; C is LC-MS response permutation testing; D is GC-MS response permutation testing. Data statistics Through LC/GC-MS technology, the intestinal tract of the S. paramamosain was analyzed, and a total of 248 differential metabolites were detected (Fig. A). Through data analysis, these differential metabolites were classified into 12 Super Classes, 51 Classes, and 80 Sub Classes (Fig. ). “Amino acids, peptides, and analogues” account for the highest proportion in the sub class, with 37 types of metabolites identified, accounting for 15% (Fig. A); In Class, metabolites with poor metabolism are mainly concentrated in “Fatty Acyls”, “Carboxylic acids and derivatives”, and “Glycerophoripids” (Fig. B); In Super Class, “Lipids and lip like molecules” and “Organic acids and derivatives” account for as much as 56% (Fig. C). Differential metabolite screening Under the log 2 FC (fold change) and -log 10 P values and VIP screening conditions, 248 differential metabolites were determined in the intestinal tract of S. paramamosain paraculata under long-term chloride saline-alkali aquaculture stress, of which 112 were up-regulated and 136 were down-regulated (Fig. ). The upregulated differential metabolites are divided into 30 classes. Mainly concentrated in “Fatty Acyls”, “Glycerophospholipids”, “Carboxylic acids and derivatives”, and “Organooxygen”. In organic oxygen compounds (Fig. A). Among them, “Fatty Acyls” has the highest number of differential metabolites, which account for 25% of the differential metabolites; It is followed by “Glycerophospholipids”, accounting for 19% (Fig. A). These major up-regulated differential metabolites are “Lipids and lips-like molecules”. The down-regulated differential metabolites were divided into 39 categories, and the most down-regulated differential metabolites were “Carboxylic acids and derivatives”. In addition, “Organic acids and derivatives” was the second largest type of differential metabolite, accounting for 24% (Fig. B). Metabolic pathway analysis Through KEGG metabolic pathway enrichment analysis, 50 metabolic pathways were identified in the C/DC group, with 11 significantly enriched ( P < 0.05 ) and 39 not significantly enriched ( P > 0.05 ). 11 significantly enriched metabolic pathways include “Metabolism of other amino acids” (“D-Amino acid metabolism”, “beta Alanine metabolism”, and “Taurine and Hypoaurine metabolism”), “Membrane Transport” (“ABC transporters”), and “Transport and catabolism” (“Autophagy other”) (Fig. ) (Table ). Based on the above analysis, it can be concluded that among the 11 significantly enriched metabolic pathways, the main ones are “Amino acid metabolism/metabolism of other amino acids” and “Lipid metabolism” (Table ). The metabolic pathways related to amino acid metabolism mainly involve amino acids such as “L-Arginine (1+)”, “L-Methionine”, “L-Histidine”, “L-Cysteine” and “L-Arginine” (Table ). The differential metabolites involved in the lipid metabolism pathway mainly include two types: “Biosynthesis of unsaturated fatty acids” and “Glycerophospholipid metabolism”, involving “Oleic Acid”, “Epa”, “Docosahexaenoic Acid”, “Cis Gondonic Acid”, “Phosphatidylcholine”, “Phosphatidylethanolamine” and “Lysophospholipid” (Table ). 

Using non targeted metabolomics analysis, the metabolic changes in the intestinal tract of the S. paramamosain under long-term chloride salt alkali water stress were studied. LC/GC-MS detected a total of 4113 metabolites. To verify the validity of the data, OPLS-DA discriminant analysis statistical method (Fig. ) was used to improve the analytical ability and effectiveness of the model, maximizing the differences between different groups within the model. According to the OPLS-DA score plot, it can be seen that there is a significant separation between the control group (CK) and the long-term chloride type saline-alkali water stress group (LSAW group) (Fig. A and B), indicating that LSAW group leads to significant changes in the metabolite profile of the S. paramamosain . Through the fitting test of the OPLS-DA model, it can be found that the explanatory power (R 2 Y (cum)) and predictive power (Q 2 (cum)) of the model for the sample are 0.999 and 0.919 (GC-MS) and 0.994 and 0.906 (LC-MS), respectively (Fig. C and D). In addition, the slope of the straight line is relatively large, and the intercept of Q 2 is -0.243 (GC-MS) and − 0.183 (LC-MS), indicating that the OPLS-DA model does not exceed the fit (Fig. ). In summary, the OPLS-DA model has good explanatory and predictive abilities, and can reflect the differences between different sample groups in real responses. The above results indicate that compared with the CK group, there were significant changes in the metabolic products of the intestinal tract in the LSAW group of S. paramamosain . A is LC-MS OPLS-DA scores plots; B is GC-MS OPLS-DA scores plots; C is LC-MS response permutation testing; D is GC-MS response permutation testing. 

Through LC/GC-MS technology, the intestinal tract of the S. paramamosain was analyzed, and a total of 248 differential metabolites were detected (Fig. A). Through data analysis, these differential metabolites were classified into 12 Super Classes, 51 Classes, and 80 Sub Classes (Fig. ). “Amino acids, peptides, and analogues” account for the highest proportion in the sub class, with 37 types of metabolites identified, accounting for 15% (Fig. A); In Class, metabolites with poor metabolism are mainly concentrated in “Fatty Acyls”, “Carboxylic acids and derivatives”, and “Glycerophoripids” (Fig. B); In Super Class, “Lipids and lip like molecules” and “Organic acids and derivatives” account for as much as 56% (Fig. C). 

Under the log 2 FC (fold change) and -log 10 P values and VIP screening conditions, 248 differential metabolites were determined in the intestinal tract of S. paramamosain paraculata under long-term chloride saline-alkali aquaculture stress, of which 112 were up-regulated and 136 were down-regulated (Fig. ). The upregulated differential metabolites are divided into 30 classes. Mainly concentrated in “Fatty Acyls”, “Glycerophospholipids”, “Carboxylic acids and derivatives”, and “Organooxygen”. In organic oxygen compounds (Fig. A). Among them, “Fatty Acyls” has the highest number of differential metabolites, which account for 25% of the differential metabolites; It is followed by “Glycerophospholipids”, accounting for 19% (Fig. A). These major up-regulated differential metabolites are “Lipids and lips-like molecules”. The down-regulated differential metabolites were divided into 39 categories, and the most down-regulated differential metabolites were “Carboxylic acids and derivatives”. In addition, “Organic acids and derivatives” was the second largest type of differential metabolite, accounting for 24% (Fig. B). 

Through KEGG metabolic pathway enrichment analysis, 50 metabolic pathways were identified in the C/DC group, with 11 significantly enriched ( P < 0.05 ) and 39 not significantly enriched ( P > 0.05 ). 11 significantly enriched metabolic pathways include “Metabolism of other amino acids” (“D-Amino acid metabolism”, “beta Alanine metabolism”, and “Taurine and Hypoaurine metabolism”), “Membrane Transport” (“ABC transporters”), and “Transport and catabolism” (“Autophagy other”) (Fig. ) (Table ). Based on the above analysis, it can be concluded that among the 11 significantly enriched metabolic pathways, the main ones are “Amino acid metabolism/metabolism of other amino acids” and “Lipid metabolism” (Table ). The metabolic pathways related to amino acid metabolism mainly involve amino acids such as “L-Arginine (1+)”, “L-Methionine”, “L-Histidine”, “L-Cysteine” and “L-Arginine” (Table ). The differential metabolites involved in the lipid metabolism pathway mainly include two types: “Biosynthesis of unsaturated fatty acids” and “Glycerophospholipid metabolism”, involving “Oleic Acid”, “Epa”, “Docosahexaenoic Acid”, “Cis Gondonic Acid”, “Phosphatidylcholine”, “Phosphatidylethanolamine” and “Lysophospholipid” (Table ). 

The intestinal tract is an important organ, and it is not only an important organ for nutrient absorption , also involved in barrier function and immune regulation. The intestinal tract is the first-line physical barrier of animals against pathogen infection . A high carbohydrate diet can damage the digestive system of Micropterus salmoides , manifested as an increase in the number of intestinal inflammatory cells and mucosal loss . When faced with cadmium stress, Procambarus clarii experiences an inflammatory response in its intestine, manifested as shedding of the peritrophic membrane, disordered arrangement and nuclear deformation in intestinal epithelial cells, dissolution of connective tissue, disruption of tight junction complexes, substantial decrease in mitochondria, and partial loss of microvilli in specific regions . In this study, LC/GC-MS was used to analyze the metabolic effects of the intestinal tract of S. paramamosain under the stress of long-term chlorinated low-salinity saline-alkaline water culture. Under the stress of long-term chlorinated low-salinity saline-alkaline aquaculture, the metabolites of “Lipids and lips-like molecules” were up-regulated the most. The up-regulation of lipid metabolites indicated that lipid storage was increased. This may increase the resistance of S. paramamosain to low-salt saline-alkali water environmental stress. It was found that the levels of phospholipids and fatty acid metabolites in the intestinal tract of Penaeus monodon were increased after exposure to Vibrio harveyi . Under low-salt stress, lipid substances in the antennal gland of the mud crabs significantly increased . Under acute low-salt stress, triglycerides and cholesterol in the hemolymph of Eriocheir sinensis were significantly increased and used as energy expenditure to cope with external environmental stress . The results of this study showed that among the up-regulated differential metabolites, “PC(P-18:1(11Z)/22:5(4Z,7Z,10Z,13Z,19Z)-O(16,17)) ” had the highest VIP value and the most prominent contribution. “PC (P - now (z)/whoever (z, z, z, 10, 13 z, z) 19 - O (16 or 17)) ” (VIP = 8.89), “PC ( P − 16:0/18:1 (z) − 2 oh (9, 10)) ” (VIP = 7.41), “PC (and (z z, 12, 15 z)/(z, z, z, 10, 13 z, z, z) 19) ” (VIP = 4.58) and “PC (P − 16:0/(5 z, 7 z, z, z, z, z) 19 - OH (4)) ” (VIP = 1.98) is higher, these contributions for the oxidation of phosphatidyl choline. Phosphatidylcholine is the main component of cell membrane and an important component of pulmonary surfactant and serum lipoprotein in vivo . Studies have shown that oxidized phospholipids can exist in vivo in many different derivatives and isomers, and the CD36 form of oxidized phosphatidylcholine accumulates in vivo and mediates the formation of macrophage foam cells, and promotes platelet hyperreactivity in hyperlipidemia through CD36 . Studies have also shown that oxidized phosphatidylcholine is very important for the exploration of novel bioactive substances as candidate biomarkers for various inflammatory diseases and common diseases . It is speculated that the intestinal inflammation will occur for a long time when the mud crab is cultured in long-term chlorinated low-salt saline water. Intestinal hyperplasia occurs under the stimulation of inflammatory factors, tissue disintegration products or some physical and chemical factors, and it is this proliferative reaction that allows the damaged tissues to be repaired to adapt to long-term environmental stress. Indole is involved in regulating intestinal immune homeostasis during disease infection . Research has shown that indole and compounds with indole rings have antibacterial, anti-inflammatory, and antioxidant effects . In mice, indole-3-carbinol was added significantly ameliorated body weight loss, colon length shortening and colonic pathological damage in colitis mice, reduced disease activity index and histological scores, as well as alleviated colonic necroptosis and inflammation . In LSAW group, “Indoacrylic Acid” (VIP = 24.09) contributed the most to the down-regulation of metabolic poor foreign bodies. Under long-term chloride low-salt saline-alkali aquaculture, the down-regulation of indole may explain the proliferation caused by long-term inflammation in the intestine, and the weakening of anti-inflammatory effects may lead to long-term inflammation and proliferation in the intestine. Among all the down-regulated metabolites, “Amino acids, peptides, and analogues” were the most down-regulated, including amino acids such as “L-Argininium (1+)”, “L-Methionine”, “L-Histidine”, “L-Cysteine”, and “L-Arginine” (Fig. B). Amino acids are the foundation of all life forms and are components of peptides and proteins, which are crucial for the survival and growth of organisms . There are also studies indicating that many organisms experience a decrease in amino acid levels after being subjected to external stress. For example, both mud crab was stressed by bicarbonate  or acute low-salt stress  and Penaeus vanname was exposed to the air exhibit this phenomenon . Therefore, the decrease in amino acids in S. paramamosain under long-term chloride type low-salt saline-alkali aquaculture stress may indicate that amino acids are used as an energy source for the immune response of mud crabs to long-term environmental stress. Through KEGG pathway enrichment analysis, it was found that the amino acid metabolism pathway was significantly enriched. Compared with CK, “D-Amino acid metabolism”, “Beta Alanine metabolism”, “Taurine and hypotaurine metabolism” and “Arginine biosynthesis” were significantly enriched in LSAW. Among the 11 significantly differentially expressed metabolic pathways, “D-Amino acid metabolism”, “Arginine biosynthesis”, “Aminoacyl tRNA biosynthesis” and “ABC transporters” are all involved in the regulation of L-Arginine metabolism (Table ). Arginine acts as a functional amino acid and performs various physiological functions in the body . For example, promoting cell proliferation, maintaining intestinal homeostasis, immune response, antioxidant activity, hormone secretion, metabolism, and regulating animal energy metabolism . The down-regulation of L-Arginine may be due to the use of energy sources for immune responses in response to external low-salt saline-alkali water stress in the S. paramamosain , which disrupts the original homeostasis in the intestine and shifts to another homeostasis for development . At the same time, this also explains the occurrence of intestinal hyperplasia to cope with external low-salt saline-alkali water stress. Through pathway enrichment analysis of differential metabolites, it was found that compared with the CK group, “Glycerophoripip metabolites” were significantly enriched. Research has shown that S. paramamosain undergo energy related metabolic regulation to cope with low-salt stress, and lipids are also an important pathway for energy production . The up-regulation of foreign substances related to “Glycophorospholipid metabolism” provides energy for the S. paramamosain to cope with low-salt saline-alkali water stress, indicating that more energy is needed to repair the intestinal barrier under low-salt saline-alkali water pressure. “Glycophospholipid metabolism” is the main pathway involved in systemic immunity and low-grade inflammation, indicating that phospholipids are potential inflammatory mediators . Previous studies have shown that phosphatidylcholine has a regulatory effect on inflammatory response and inhibits inflammation under various conditions . Therefore, under long-term saline-alkali water aquaculture, the enhancement of glycerophospholipids in the intestine can alleviate the inflammatory response in the intestinal tract of the S. paramamosain . 

Under long-term chloride type low-salt saline-alkali water stress, the increase in lipids in the intestinal tract of the S. paramamosain provides energy for resisting external low-salt saline-alkali water stress; The downregulation of amino acids is used as an energy source for immune response in response to external low-salt saline-alkali water stress in mud crabs. The downregulation of indole indicates that the intestinal immune homeostasis is disrupted and inflammation occurs in the intestine. The above energy sources can better repair the intestinal barrier. Under repeated stimulation, the intestine proliferates to cope with long-term external stress, and the intestine shifts to another homeostasis for development. The results of this study have provided a deeper understanding of the cultivation technology of S. paramamosain in saline-alkali soil, and have important reference and practical significance for the cultivation of S. paramamosain in saline-alkali soil. 

Experimental animal The experimental S. paramamosain were purchased from local market of Sanmen Bay, Zhejiang, China (E: 121°52’53”; N: 29°1’29”). Firstly, mud crabs (15 ± 2 g) with uniform individuals, complete limbs and good vitality were cultured in the breeding pond in Sanmen, Zhejiang Province, with a salinity of 18‰ (CK group). Secondly, another part of the mud crabs were desalinated to 3‰ (After temporary cultivation, the salinity will be reduced from 22‰ to 16‰, followed by 12‰, 10‰, 8‰, 6‰, 5‰, 4‰, and 3‰. During this period, the desalination interval will be 6 h acclimatization.). After the salinity was desalinated to 3%, the mud crabs were packed and transported by truck to Rui’an, Zhejiang Province (LSAW group). Mud crabs with uniform individuals weight (15 ± 2 g), complete limbs and good vitality were selected and then cultured in a breeding pond with a salinity of 3% in Rui’an, Zhejiang. The total alkalinity, salinity and pH of CK group were 120 mg/L, 18‰ and 8.5, respectively. The ionic composition of CK group was 282 mg/L K + , 300 mg/L Ca 2+ and 936 mg/L Mg 2+ . The total alkalinity, salinity and pH of LSAW group were 260 mg/L, 3‰ and 8.4, respectively. The ionic composition of LSAW group was 120 mg/L K + , 140 mg/L Ca 2+ and 137 mg/L Mg 2+ (Table ). The size of the pond was 5 mu (667 m 2 ), the depth was 2 m, and each pond was set up with 2 oxygen pumps, and the water change was 1/4 per month. Sample extraction After 90 days of breeding, mud crabs weight 240 ± 20 g were selected for sampling. Took 5 mud crabs from the aquaculture ponds in Sanmen Bay and Rui’an respectively, and analyzed their intestines. During sampling, the mud crab was immersed in ice water until anesthetized, and then dissected immediately . The intestinal tissue of the mud crab was isolated with sterile tweezers and placed in a cryostorage tube. They were then immediately frozen in liquid nitrogen and stored at -80 ℃. Finally, metabolomics analysis was performed ( n = 5). Sample preparation All extraction reagents are pre cooled at -20 ℃ before use. Weigh 30 mg of sample into a 1.5 mL centrifuge tube, add two small steel balls and 600 µL of methanol water (V: V = 4:1, containing mixed internal standard, 4 µg/mL), place in a -40 °C refrigerator for 2 min, then grind in a grinder (60 Hz, 2 min), extract with ice water bath ultrasound for 10 min, let it stand overnight at -40 °C, and centrifuge at low temperature for 10 min (13201 xg, 4 °C). Use a syringe to aspirate 150 µL of the supernatant, filter it using a 0.22 μm organic phase pinhole filter, transfer it to an LC injection vial, and store it at -80 °C until LC-MS analysis is performed. At the same time, take 150 µL of the supernatant and transfer it into a glass derivatization bottle. Use a centrifugal concentration dryer to evaporate the sample. Add 80 µL of methoxyamine hydrochloride pyridine solution (15 mg/mL) to the glass derivatization bottle and shake it in a culture box at 37 °C for 60 min for oximation reaction. After removing the sample, add 50 µL of BSTFA derivatization reagent and 20 µL of n-hexane. Add 10 µL of 10 internal standards (C8/C9/C10/C12/C14/C18/C20/C22/C24, all in chloroform configuration) and react at 70 °C for 60 min. Take out the sample and let it stand at room temperature for 30 min for GC-MS metabolomics analysis. Quality control samples (QC) are prepared by mixing equal volumes of extracts from all samples. Liquid chromatography-mass spectrometry analysis The chromatography was performed on ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm) in positive and negative modes. The gradient elution system consists of (A) water (containing 0.1% formic acid, v/v) and (B) acetonitrile (containing 0.1% formic acid, v/v) using the following gradients: 0.01 min, 5% B; 2 min, 5% B; 4 min, 30% B; 8 min, 50% B; 10 min, 80% B; 14 min, 100% B; 15 min, 100% B; At 15.1 min, 5% and 16 min, the 5% flow rate was 0.35 mL/min and the column temperature was 45℃. All samples were kept at 10 ℃ during analysis. The sample size was 3 µL. Spray voltage, 3800 V and 3200 V(−); Sheath gas flow, 35 arbitrary units; Auxiliary gas flow, 8 arbitrary units; Capillary temperature: 320℃; Aux gas heater temperature, 350℃; s lens RF level, 50. Gas chromatography-mass spectrometry analysis conditions The derivatives were isolated using DB-5MS fused quartz capillary column. The carrier gas was high purity helium (purity not less than 99.999%), the flow rate was 1.0 mL/min, and the temperature of the inlet was 260 ℃. The sample size was 1 µL without shunt. Programmed temperature rise: the initial temperature of the column temperature box is 60 ℃ and maintained for 0.5 min; Heat up to 125℃ at 8 ℃/min; 8 ℃/min to 210 ℃; 15 ℃/min to 270 ℃; Temperature rise from 20 ℃/min to 305 ℃ for 5 min. Electron bombardment ion source (EI), ion source temperature 230 ℃, quadrupole temperature 150 ℃, electron energy 70 eV. Scanning mode is full scanning mode (SCAN), quality scanning range: m/z 50–500. Data analysis Using orthogonal partial least squares analysis (OPLS-DA) to filter out noise unrelated to classification information, improving the analytical ability and effectiveness of the model, and maximizing the differences between different groups within the model. To prevent overfitting of the model, seven-fold cross validation and 200 response permutation testing (RPT) were used to evaluate the quality of the model. Differential metabolites with VIP value greater than 1.0 and p value less than 0.05 were selected. Using KEGG compound data ( http://www.kegg.jp/kegg/compound/ ) Annotate identified metabolites and then map the annotated metabolites to the KEGG pathway database ( http://www.kegg.jp/kegg/pathway.html ). For a given list of metabolites, use the p-value of hypergeometric test to identify significantly enriched pathways. 

The experimental S. paramamosain were purchased from local market of Sanmen Bay, Zhejiang, China (E: 121°52’53”; N: 29°1’29”). Firstly, mud crabs (15 ± 2 g) with uniform individuals, complete limbs and good vitality were cultured in the breeding pond in Sanmen, Zhejiang Province, with a salinity of 18‰ (CK group). Secondly, another part of the mud crabs were desalinated to 3‰ (After temporary cultivation, the salinity will be reduced from 22‰ to 16‰, followed by 12‰, 10‰, 8‰, 6‰, 5‰, 4‰, and 3‰. During this period, the desalination interval will be 6 h acclimatization.). After the salinity was desalinated to 3%, the mud crabs were packed and transported by truck to Rui’an, Zhejiang Province (LSAW group). Mud crabs with uniform individuals weight (15 ± 2 g), complete limbs and good vitality were selected and then cultured in a breeding pond with a salinity of 3% in Rui’an, Zhejiang. The total alkalinity, salinity and pH of CK group were 120 mg/L, 18‰ and 8.5, respectively. The ionic composition of CK group was 282 mg/L K + , 300 mg/L Ca 2+ and 936 mg/L Mg 2+ . The total alkalinity, salinity and pH of LSAW group were 260 mg/L, 3‰ and 8.4, respectively. The ionic composition of LSAW group was 120 mg/L K + , 140 mg/L Ca 2+ and 137 mg/L Mg 2+ (Table ). The size of the pond was 5 mu (667 m 2 ), the depth was 2 m, and each pond was set up with 2 oxygen pumps, and the water change was 1/4 per month. 

After 90 days of breeding, mud crabs weight 240 ± 20 g were selected for sampling. Took 5 mud crabs from the aquaculture ponds in Sanmen Bay and Rui’an respectively, and analyzed their intestines. During sampling, the mud crab was immersed in ice water until anesthetized, and then dissected immediately . The intestinal tissue of the mud crab was isolated with sterile tweezers and placed in a cryostorage tube. They were then immediately frozen in liquid nitrogen and stored at -80 ℃. Finally, metabolomics analysis was performed ( n = 5). 

All extraction reagents are pre cooled at -20 ℃ before use. Weigh 30 mg of sample into a 1.5 mL centrifuge tube, add two small steel balls and 600 µL of methanol water (V: V = 4:1, containing mixed internal standard, 4 µg/mL), place in a -40 °C refrigerator for 2 min, then grind in a grinder (60 Hz, 2 min), extract with ice water bath ultrasound for 10 min, let it stand overnight at -40 °C, and centrifuge at low temperature for 10 min (13201 xg, 4 °C). Use a syringe to aspirate 150 µL of the supernatant, filter it using a 0.22 μm organic phase pinhole filter, transfer it to an LC injection vial, and store it at -80 °C until LC-MS analysis is performed. At the same time, take 150 µL of the supernatant and transfer it into a glass derivatization bottle. Use a centrifugal concentration dryer to evaporate the sample. Add 80 µL of methoxyamine hydrochloride pyridine solution (15 mg/mL) to the glass derivatization bottle and shake it in a culture box at 37 °C for 60 min for oximation reaction. After removing the sample, add 50 µL of BSTFA derivatization reagent and 20 µL of n-hexane. Add 10 µL of 10 internal standards (C8/C9/C10/C12/C14/C18/C20/C22/C24, all in chloroform configuration) and react at 70 °C for 60 min. Take out the sample and let it stand at room temperature for 30 min for GC-MS metabolomics analysis. Quality control samples (QC) are prepared by mixing equal volumes of extracts from all samples. 

The chromatography was performed on ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm) in positive and negative modes. The gradient elution system consists of (A) water (containing 0.1% formic acid, v/v) and (B) acetonitrile (containing 0.1% formic acid, v/v) using the following gradients: 0.01 min, 5% B; 2 min, 5% B; 4 min, 30% B; 8 min, 50% B; 10 min, 80% B; 14 min, 100% B; 15 min, 100% B; At 15.1 min, 5% and 16 min, the 5% flow rate was 0.35 mL/min and the column temperature was 45℃. All samples were kept at 10 ℃ during analysis. The sample size was 3 µL. Spray voltage, 3800 V and 3200 V(−); Sheath gas flow, 35 arbitrary units; Auxiliary gas flow, 8 arbitrary units; Capillary temperature: 320℃; Aux gas heater temperature, 350℃; s lens RF level, 50. 

The derivatives were isolated using DB-5MS fused quartz capillary column. The carrier gas was high purity helium (purity not less than 99.999%), the flow rate was 1.0 mL/min, and the temperature of the inlet was 260 ℃. The sample size was 1 µL without shunt. Programmed temperature rise: the initial temperature of the column temperature box is 60 ℃ and maintained for 0.5 min; Heat up to 125℃ at 8 ℃/min; 8 ℃/min to 210 ℃; 15 ℃/min to 270 ℃; Temperature rise from 20 ℃/min to 305 ℃ for 5 min. Electron bombardment ion source (EI), ion source temperature 230 ℃, quadrupole temperature 150 ℃, electron energy 70 eV. Scanning mode is full scanning mode (SCAN), quality scanning range: m/z 50–500. 

Using orthogonal partial least squares analysis (OPLS-DA) to filter out noise unrelated to classification information, improving the analytical ability and effectiveness of the model, and maximizing the differences between different groups within the model. To prevent overfitting of the model, seven-fold cross validation and 200 response permutation testing (RPT) were used to evaluate the quality of the model. Differential metabolites with VIP value greater than 1.0 and p value less than 0.05 were selected. Using KEGG compound data ( http://www.kegg.jp/kegg/compound/ ) Annotate identified metabolites and then map the annotated metabolites to the KEGG pathway database ( http://www.kegg.jp/kegg/pathway.html ). For a given list of metabolites, use the p-value of hypergeometric test to identify significantly enriched pathways. 

