Effect of Different Diets on the Hepatopancreatic Proteomes of Chinese Mitten Crab (Eriocheir Sinensis)

Aquatic plants and freshwater snails are important natural food sources of Eriocheir sinensis. The effects of these two kinds of natural food sources on the growth and development of Eriocheir sinensis were studied by determining the hepatopancreatic proteomes of three crab groups, namely, crabs fed with aquatic plants combined with freshwater snails (group A), crabs fed with aquatic plants only (group B), and crabs fed with freshwater snails only (group C), with tandem mass tag technology. Results showed 110 differentially expressed proteins between groups A and B, among which 78 were up-regulated and 32 were down-regulated in group A. Meanwhile, 9 proteins were up-regulated and 14 proteins were down-regulated in group A relative to those in group C. The proteins related to molting and growth that were differentially expressed between groups A and B were up-regulated in group A. These proteins included cryptocyanin and cuticle protein CBM. The immunity-related proteins, such as mannosyl-oligosaccharide glucosidase and glutathione peroxidase, that were differentially expressed between groups A and C and were up-regulated in group A. These results indicated that freshwater snails might promote the growth and development of E. sinensis to a certain extent, and aquatic plants might play an important role in the immunity of E. sinensis. Our study provides a theoretical basis for the practice of “planting grass and throwing snails” in the green ecological culture of E. sinensis.


Introduction
Chinese mitten crab (Eriocheir sinensis), commonly known as river crab, is an important economic aquaculture species in China (1). The planting of aquatic plants has become one of the key factors for the success of crab culture. Aquatic plants can not only regulate the pH of water and provide shelter to crabs, they can also be used as food sources by crabs due to their rich nutrient contents (2,3). Several researchers have indicated that aquatic plants are bene cial for crab growth and can improve the nutritional quality of the edible parts of crabs. Crabs ingest a certain amount of aquatic plants to meet their nutritional needs even when receiving su cient feed (4). Freshwater snails are a high-quality natural feed for crabs, and snail feeding in the process of crab culture can increase breeding yield and improve quality (5,6). Snails, as an animal feed, can increase the content of animal protein; this effect has a certain in uence on crab growth (7).
The growth and development of organisms are closely related to food sources. Different food sources cause changes in the composition of proteins in tissues and organs; affect biological processes, such as digestion and absorption, energy metabolism, and immune response; and further affect the growth and development of organisms (8,9). Comparing the liver proteomes of rats fed with animal protein with those of rats fed with plant protein revealed that the two groups exhibited drastic changes in their protein expression pro les and considerably different amino acid metabolism and fatty acid metabolism (10). The content of proteins related to lipid, carbohydrate, and amino acid metabolism changed in the livers of Oreochromis niloticus fed with diets containing different nutrients, and the immune systems of the test organisms also changed (11). Replacing dietary sh oil with linseed oil, resulted in considerable changes in the protein expression pro le in the hepatopancreas of E. sinensis; subsequently, the capability of this crab species to adapt to the environment was also altered (12). Food sources can obviously affect the protein composition of organisms and then further affect the growth and development of organisms.
Aquatic plants and freshwater snails play an important role in the growth and development of crabs as two kinds of important natural food. However, research on how aquatic plants and snails affect the growth and development of crabs remains scant.
A comprehensive analysis of the composition and dynamics of proteins offers important insights into the roles of aquatic plants and snails in the process of crab culture. Therefore, in this study, three diverse feed types were provided as daily crab diets: freshwater snail (Sinotaia quadrata); waterweed plants (Elodea canadensis); and a mixed diet of S. quadrata and E. canadensis. Then, the protein pro les of crabs under the three different feed types were determined and compared to investigate the effect of aquatic plants and snails on crab growth and development.

Sample collection and ethics statement
Juvenile crabs (approximately 7.5 g) with similar growth conditions were collected from the aquatic animal germplasm resource station of Shanghai Ocean University. They were fed in a circulating water system for 7 days to adapt to the environment. Then, the crabs were randomly divided into three groups and fed as follows: Group A was fed with a mixed diet of S. quadrita and E. canadensis, group B was fed with E. canadensis only, and group C was fed with S. quadrita only. All the crabs were reared in the "Crab Dragon Palace" in the same environment. The water temperature was maintained at 26 ℃ ± 2 ℃, and the three groups were fed with the same amount of food at 9:00 every day. When the crabs grew to the early stage of molting, their hepatopancreas tissues were collected. Three biological repeats were set for each group. Then, the tissues were quickly frozen in liquid nitrogen and stored in a −80 ℃ refrigerator.
The whole process follows the institutional animal care and use committee of Shanghai Ocean University (Shanghai, China).

Protein extraction and quality control
The collected hepatopancreatic tissues were removed from −80 °C refrigerator and homogenized.
Approximately 50 mg of minced tissue was mixed with 500 µl of RIPA lysate (PMSF was added before use). Subsequently, the homogenate was incubated in an ice bath for 30 min. Centrifugation was performed at 14 000 g for 10 min at 4 °C. The supernatant was collected in a new tube. Protein concentration was measured with a Pierce BCA protein assay kit in accordance with instructions (Thermo sher, USA), and protein quality was tested through SDS-PAGE gel electrophoresis.
Protein alkylation, trypsin enzymatic hydrolysis, and TMT tagging The proteins were alkylated in accordance with Randall (13), and the lter-aided proteome preparation method was used for protease hydrolysis (14). The trypsin enzyme was added on the basis of the ratio of protein: enzyme = 40:1. The mixture was placed at 37 ℃ overnight. Then, the peptide segment was desalted and lyophilized. A total of 100 µg of protein was taken from each sample for TMT labeling. The labeling steps are as follows: First, the temperature of the TMT reagent was allowed to recover to room temperature. Then, acetonitrile was added to the sample, and the sample was centrifuged at low speed with a vortex. Second, the sample was mixed with TMT reagent, incubated at room temperature for 2 h, and then mixed with hydroxylamine. The mixture was reacted at room temperature for 15 min. Finally, the same amount of labeled substances was mixed in a tube and drained with a vacuum concentrator.

HPLC fractionation and LC-MS/MS analysis
Polypeptide samples were redissolved with UPLC loading buffer, and a reverse phase C18 column was used to separate the high pH liquid phase. A total of 20 fractions were collected and merged into 10 fractions in accordance with peak type and time. After vacuum centrifugation and concentration, the mass spectrometry sample was dissolved with the loading buffer solution for mass spectrometry. The mass spectrometry conditions were as follows: The data acquisition software was Thermo Xcalipur 4.0 (Thermo, USA). The chromatographic instrument was Easy NLC 1200 (Thermo, USA), and the mass spectrometer was Q_Exactive HF-X (Thermo, USA). The chromatographic separation time was 120 min, the ow rate was 300 nl/min, the scanning range of MS was 350-1300 m/z, and the acquisition mode was DDA.

Bioinformatic analysis
ProteomeDiscovererTM software 2.4 was used to search NCBI and the Uniprot database to identify and quantify proteins. Proteins with fold change (FC) < 0.667, FC >1.5, and P < 0.05 were considered as differentially expressed proteins. Differentially expressed proteins were subjected to GO and KEGG enrichment analysis by using the software implemented in Majorbio I-Sanger Cloud Platform with corrected P < 0.05.

Results
Overview of total identi ed proteins After submitting the original data le of the mass spectrometer off machine to the Proteome Discoverer server, 358,336 secondary spectra were obtained, 67,127 spectra were matched, and 24,744 peptide fragments and 9,959 proteins were identi ed (Fig. 1). After the functional annotation of the identi ed proteins, 4,277 annotated proteins were obtained. Among these proteins, 2,532 proteins were annotated in GO enrichment, accounting for 59.2% of the total annotated proteins, and 3,041 were annotated KEGG pathway, accounting for 71.1% of the total annotated proteins (Table 1). These results indicated that the proteomic data of this study were reliable. Bioinformatic analysis of differentially expressed proteins The hepatopancreatic proteomics of the three groups were compared and analyzed. A total of 323 differentially expressed proteins were identi ed with the statistical thresholds of P < 0.05, FC > 1.5, or FC < 0.67. Compared with those in group B, 78 proteins were highly expressed and 32 proteins were expressed at low levels in group A. Compared with those in group C, 9 were highly expressed and 14were expressed at low levels in group A (Fig. 2).
GO enrichment analysis indicated that the differentially expressed proteins between groups A and B were mainly enriched in hydrolase activity, deacetylation, lipoprotein metabolism, and galactosidase activity.
The differentially expressed proteins between groups A and C were mainly enriched in oxidative stress reaction and amino acid metabolism (Fig. 3). KEGG database was used to analyze the enrichment of differentially expressed proteins in metabolic pathways. The results showed that the differentially expressed proteins between groups A and B were mainly enriched in lysosomes, sphingolipid metabolism, and polysaccharide degradation pathways, whereas the differentially expressed proteins between groups A and C were mainly enriched in metabolic pathways related to infection (Fig. 4).

Differentially expression proteins between groups
The proteomes of groups A and B were compared and analyzed. The proteins that were highly expressed in group A included cryptocyanin, cuticle protein, solute carrier family 35 member F6, programmed cell death protein, broblast growth factor receptor 3. The proteins expressed at low levels in group A were ataxin-2, metalloreductase, pancreatic lipase-related protein 2, and arylsulfatase A ( Table 2). The proteomes of groups A and C were also compared and analyzed. Mannosyl oligosaccharide glucosidase, glutathione peroxidase 2, calreticulin were among the proteins that were highly expressed in group A relative to group C. The proteins that were expressed at low levels in group A were myosin and Rhoassociated protein kinase 2 (Table 3).

Discussion
Aquatic plants and freshwater snails are important natural food sources for crabs and have a direct effect on crab growth and development (15,16). This study was conducted to investigate the differences in the hepatopancreatic proteomes of crabs under three different feeding methods: aquatic plants combined with freshwater snails (group A), aquatic plants only (group B), and freshwater snails only (group C). The results showed differences in the protein expression pro les in the hepatopancreas of crabs among the three groups. A total of 110 proteins were differentially expressed between groups A and B, whereas only 23 proteins were differentially expressed between groups A and C. Freshwater snails might affect the protein expression pro les of crabs more than aquatic plants.
Among the differentially expressed proteins between groups A and B, the proteins with high expression levels and the most signi cant differences in group A were cryptocyanin and cuticle protein CBM. Cryptocyanin is an important member of the hemocyanin gene family and a crustacean molting protein that is closely related to molting and plays an important role in new exoskeleton formation after molting (17,18). Cuticle protein is an important component of the crab exoskeleton. During molting, the old epidermis falls off, and a new epidermis form. Cuticle protein plays a vital role in the formation of the epidermis during molting (19). Cryptocyanin and cuticle protein expression levels were signi cantly higher in crabs fed with aquatic plants and freshwater snails than in those fed with aquatic plants only, indicating that the molting frequency of crabs fed with aquatic plants and freshwater snails might be accelerated; this result was consistent with our previous research results showing that the molting rate of crabs fed with aquatic plants and freshwater snails is signi cantly faster than that of crabs fed with aquatic plants only (20). The results of this study showed that the addition of snails to crab diets could affect the expression of molting-related proteins and further affect the molting rate. A large number of proteins related to cell proliferation and growth were highly expressed in crabs fed with aquatic plants and freshwater snails; these proteins included solute carrier family 35 member F6, programmed cell death protein 2, UCN-45 protein homolog A, and broblast growth factor receptor 3 (21, 22) ( Table 2). The high expression of these proteins leads to the increase in cell number and volume and further affects crab growth and development. Therefore, crabs fed with aquatic plants and freshwater snails grew signi cantly faster than crabs fed with aquatic plants.
Among the proteins that were differentially expressed between groups A and C, those that were highly expressed in group A were mannosyl oligosaccharide glucosidase, glutathione peroxidase-2, and calreticulin. Mannosyl oligosaccharide glucosidase is involved in the metabolism of mannan oligosaccharides and can improve immunity in Litopenaeus vannamei (23,24). Glutathione peroxidase plays an important role in immune defense against pathogen infection in invertebrates. Research on Haliotis discus, Chlamys farreri, L. vannamei, and Fenneropenaeus chinensis has shown that glutathione peroxidase is involved in the immune regulation process (25)(26)(27)(28). Calreticulin is a highly conserved calcium binding protein, which is an immune-related protein in vertebrates and invertebrates. Studies on Patinopecten yessoensis, Sebastes schlegeli, and Tilapia niloticus all showed that calreticulin is involved in immune function (29)(30)(31). The levels of these proteins were higher in crabs fed with aquatic plants and freshwater snails than in crabs fed with only freshwater snails, suggesting that aquatic plants might affect crab immunity; moreover, these results were consistent with previous results that submerged plants in the diet can enhance crab immunity (32,33).
In conclusion, diets containing aquatic plants can enhance crab immunity, while those containing freshwater snails can promote crab growth and molting. A mixed diet containing aquatic plants and freshwater snails is the best choice for crabs. The results of this work provide a theoretical basis for the practice of "planting grass and throwing snails" in green crab ecological culture.

Declarations
Ethics approval and consent to participate Not applicable.