Application of arginine in promoting micropterus salmoides to resist nocardia seriolae infection
By adding arginine to the feed of largemouth bass, the mTORC1 pathway is activated, antibody secretion is promoted, and cell death is inhibited, thus solving the problem of prevention and control of nocardiosis in largemouth bass and improving survival rate and disease resistance.
Patent Information
- Application Number
- CN202511136956.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-14
- Publication Date
- 2025-11-11
AI Technical Summary
In largemouth bass farming, nocardiac disease caused by Nocardia amberjack leads to high mortality rates, and existing technologies are insufficient to effectively prevent and control it, thus affecting the high-quality development of the aquaculture industry.
Adding arginine to the feed of largemouth bass activates the mTORC1 pathway, promotes antibody secretion, inhibits cell death, maintains tissue integrity, and enhances disease resistance.
It significantly improves the mTORC1 pathway activity, antibody secretion level, cell survival rate and tissue structure integrity of largemouth bass, thereby enhancing resistance to Nocardia amberjack and improving survival rate.
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Figure CN120919104A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of functional feed additive regulation technology for fish disease resistance, specifically involving the application of arginine in promoting antibody secretion and inhibiting cell death to resist Nocardia auricula infection. Background Technology
[0002] Largemouth bass ( Micropterus salmoides The largemouth bass, commonly known as the California bass, belongs to the order Perciformes, family Solariidae, and genus Perciformes. It is a carnivorous fish native to the Mississippi River basin in North America. Introduced to my country in the 1980s, its aquaculture production has increased year by year with breakthroughs in fry breeding and artificial feed technology. Currently, its production ranks only after the four major freshwater fish species and tilapia, earning it the reputation of "China's fifth major freshwater fish." In recent years, with the rise of intensive and high-density aquaculture, outbreaks of bacterial and viral diseases have occurred, causing significant economic losses for fish farmers. Among these, Nocardia amberjack (Nocardia purpurea) is a major cause of disease outbreaks. Nocardia seriolae Nocardiac disease, caused by [unspecified pathogen], leads to high mortality rates in farmed largemouth bass, seriously threatening the high-quality development of the largemouth bass industry. Therefore, researching effective strategies for the prevention and control of nocardiac disease is a crucial issue that the largemouth bass farming industry urgently needs to address. Improving disease resistance through nutritional regulation and other means is of great significance and value.
[0003] Unlike mammals, fish cannot synthesize enough arginine to meet their own needs, making arginine one of the essential amino acids for fish. Studies have shown that arginine also participates in various cellular processes as a functional amino acid. It serves as a substrate for the synthesis of many bioactive metabolites, including nitric oxide (NO), creatine, and polyamines, and plays a regulatory role in the fish's immune response. Arginine metabolism in fish undergoes remodeling during pathogen infection, and supplemental arginine can regulate both innate and adaptive immune responses in fish, including modulating inflammatory responses, promoting phagocytosis, inhibiting leukocyte apoptosis, and enhancing disease resistance. Furthermore, in juvenile grass carp, arginine can prevent copper-induced gill tissue damage and cell death, and can remodel fish nutrient metabolism to improve hypoxia-induced damage, thereby reducing environmental stress. Summary of the Invention
[0004] The purpose of this invention is to provide the application of arginine in enhancing the resistance of largemouth bass to Nocardia amberjack infection. Through research, this invention has found that arginine has functions such as activating the largemouth bass mTORC1 pathway, promoting antibody secretion, inhibiting cell death, maintaining tissue structural integrity, and resisting Nocardia amberjack infection. Therefore, it can be used as an immune enhancer to improve the activity of the largemouth bass mTORC1 pathway, antibody secretion, cell survival and tissue structural integrity, and resistance to Nocardia amberjack infection, demonstrating significant application value.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides the application of arginine in activating the mTORC1 pathway of largemouth bass to promote antibody secretion in order to resist Nocardia amber infection.
[0006] Furthermore, the indicators of the largemouth bass's resistance to Nocardia amberjack infection include: mTORC1 pathway activity, serum IgM antibody secretion level, cell death, tissue structure, and survival rate.
[0007] Furthermore, the arginine acid activates the mTORC1 signaling pathway in largemouth bass.
[0008] Furthermore, the liver of the largemouth bass after arginine addition was subjected to combined transcriptomic and metabolomics analysis, which identified that related genes and metabolites in the mTORC1 signaling pathway were significantly enriched in KEGG. Combined with Western blot analysis, it was found that arginine addition significantly increased the phosphorylation level of key proteins S6 and 4EBP1 in the mTORC1 signaling pathway, thereby activating the mTORC1 signaling pathway in the largemouth bass.
[0009] Furthermore, the arginine promoted the secretion level of IgM in the serum of largemouth bass.
[0010] Furthermore, the arginine-added largemouth bass had a serum IgM content of 14000 µg / mL after Nocardia bacillus infection, which was significantly higher than the serum IgM level of the control group (6000 µg / mL), indicating that arginine promoted the secretion of IgM in the serum of largemouth bass.
[0011] Furthermore, the arginine inhibits hepatocyte death mediated by excessive activation of the NLRP3 inflammasome in largemouth bass.
[0012] Furthermore, the largemouth bass showed significantly increased cell death and NLRP3 inflammasome-related protein expression levels in the liver after Nocardia amberjack infection, while the addition of arginine significantly inhibited cell death mediated by excessive activation of the NLRP3 inflammasome induced by bacterial infection.
[0013] Furthermore, the arginine improves the tissue structure of the liver, head kidney, and body kidney in largemouth bass.
[0014] Furthermore, the liver, head kidney, and body kidney tissues of the largemouth bass showed signs of cell vacuolar degeneration and cell membrane rupture, granulomatous nodules, epithelial necrosis, and connective tissue hyperplasia after infection with Nocardia auricula-judae. However, the addition of arginine slowed down the rupture of liver cell membranes, significantly reduced the size of granulomatous nodules in the head kidney, and significantly improved the integrity of the renal tubular epithelial structure in the body kidney.
[0015] Furthermore, the arginine improves the survival rate of largemouth bass infected with Nocardia amberjack.
[0016] Furthermore, the survival rate of largemouth bass infected with Nocardia amberjack after the addition of arginine (53.12%) was significantly higher than that of the control group (43.75%), indicating that arginine improves the survival rate of largemouth bass infected with Nocardia amberjack.
[0017] The present invention also provides a functional feed additive for largemouth bass resistant to Nocardia amberjack, the additive comprising arginine.
[0018] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention investigates the regulatory role of arginine on the resistance of largemouth bass to Nocardia amberjack infection by feeding juvenile largemouth bass with arginine as a functional feed additive. This includes nutrient metabolic remodeling and mTORC1 pathway activity, serum IgM antibody secretion levels, NLRP3 inflammasomes and cell death, tissue structure, and survival rate. The study found that arginine can promote the secretion of more IgM from B cells into the serum by activating mTORC1 pathway activity, thereby achieving antigen neutralization and phagocytosis. Simultaneously, arginine can also alleviate tissue damage caused by Nocardia amberjack infection by inhibiting cell death mediated by excessive activation of NLRP3 inflammasomes, ultimately improving the survival rate of largemouth bass after Nocardia amberjack infection. Therefore, arginine can be used as a functional feed additive to improve the resistance of largemouth bass to Nocardia amberjack infection and enhance the production performance of largemouth bass in intensive aquaculture, which is of great significance for promoting the high-quality development of largemouth bass farming. Attached Figure Description
[0019] Figure 1 This is a diagram showing the combined analysis results of the liver transcriptome and metabolome of largemouth bass after arginine feeding in Example 1 of the present invention; Figure 2 This is a Western blot image showing the protein blotting results of mTORC1 signaling pathway-related proteins in the liver of largemouth bass after arginine feeding in Example 1 of this invention. Figure 3 This is a graph showing the secretion levels of IgM antibodies in the serum of largemouth bass in different treatment groups before and after Nocardia amberjack infection in Example 1 of the present invention. Figure 4 This is a Western blot image showing the hepatocyte death and NLRP3 inflammasome-related protein in different treatment groups before and after Nocardia amberjack infection in Example 1 of the present invention. Figure 5 The images show the histological structures of the liver, head kidney, and body kidney of largemouth bass in different treatment groups before and after Nocardia amberjack infection in Example 1 of this invention.
[0020] Figure 6This is a graph showing the survival rate of largemouth bass in different treatment groups after infection with Nocardia amberjack in Example 1 of the present invention. Detailed Implementation
[0021] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0022] Example 1: Study on the ability of arginine to promote the resistance of largemouth bass to Nocardia amberjack infection. 1. Test materials Healthy, uniformly bred largemouth bass juveniles were selected from the same batch and cultured in indoor experimental tanks. They were divided into two groups: a control group (Control, C) and an arginine group (Arginine, A). Each group had three parallel tanks. Fish were fed twice daily, at 09:00 and 18:00. The control group received standard largemouth bass feed, while the arginine group received an additional 0.5% arginine supplement. After 8 weeks of continuous culture, 6 fish from each group were randomly selected for sampling. The remaining fish were then intraperitoneally injected with 100 µL of Nocardia amberjack (1*10⁻⁶). 6 A challenge experiment was conducted using samples (CFU / mL) at different time points after infection, and mortality was recorded. Collected tissue samples were fixed in paraformaldehyde and stored at -80°C for subsequent analysis.
[0023] 2. Arginine regulates the nutritional metabolism and mTORC1 pathway of largemouth bass. To investigate the regulatory effects of arginine treatment on the nutritional metabolism and related signaling pathways of largemouth bass, transcriptomic and metabolomics analyses were conducted after an 8-week rearing period. The specific experimental steps are as follows: (1) Transcriptome sequencing 1) RNA Extraction and Quality Control: Total RNA was extracted from largemouth bass tissue using the TRIZOL method. The A260 / A280 absorbance ratio of the RNA samples was detected using a Nanodrop ND-2000 (Thermo Scientific, USA), and the RIN value of the RNA was determined using an Agilent Bioanalyzer 4150 (Agilent Technologies, CA, USA). Only RNA that passed the quality control was used for library construction.
[0024] 2) Sequencing: PE libraries were prepared according to the ABclonal mRNA-seq Lib Prep Kit (ABclonal, China) instructions. mRNA was purified from 1 μg of total RNA using oligo(dT) magnetic beads and then fragmented in ABclonal First StrandSynthesis Reaction Buffer. Subsequently, using the mRNA fragments as templates, the first strand of cDNA was synthesized using random primers and reverse transcriptase (RNase H), followed by the synthesis of the second strand of cDNA using DNA polymerase I, RNase H, buffer, and dNTPs. Adapter sequences were ligated into the synthesized double-stranded cDNA fragments for PCR amplification. The PCR products were purified, and library quality was assessed using an Agilent Bioanalyzer 4150. Finally, sequencing was performed using the NovaSeq 6000 sequencing platform with PE150 reads.
[0025] 3) Quality assessment of sequencing data: The raw data was filtered using the FASTP software (https: / / github.com / OpenGene / fastp) to obtain high-quality sequencing data, which was then statistically analyzed and quality assessed again. The quality-controlled raw data was aligned with the reference genome, and the alignment results of this transcriptome sequencing were assessed using the HiSat2 software (http: / / ccb.jhu.edu / software / hisat2 / index.shtml).
[0026] 4) Analysis of differential gene expression among different samples: RSEM software (http: / / deweylab.github.io / RSEM / ) was used to quantitatively analyze the expression levels of genes and transcripts, which was used for subsequent analysis of differential gene expression among different samples. DESeq2 software (http: / / bioconductor.org / packages / stats / bioc / DESeq2) was used to analyze differential gene expression among samples and identify differentially expressed genes (DEGs). The screening criteria for significantly differentially expressed genes were: FDR < 0.05 and |log2FC| ≥ 1.
[0027] (2) Metabolomics sequencing High-throughput targeted metabolomics analysis of largemouth bass liver tissue (5 replicates per group) was performed using H650 high-throughput sequencing technology.
[0028] 1) Sample extraction: After the sample was slowly thawed at 4℃, an appropriate amount of sample was added to a pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v), vortexed, sonicated at low temperature for 30 min, allowed to stand at -20℃ for 10 min, centrifuged at 14000 g at 4℃ for 20 min, the supernatant was collected and vacuum dried, and 100 μL of acetonitrile aqueous solution (acetonitrile:water = 1:1, v / v) was added to redissolve the sample for mass spectrometry analysis, vortexed, centrifuged at 14000 g at 4℃ for 15 min, and the supernatant was injected for analysis.
[0029] 2) Chromatographic separation: Samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) system with HILIC and C18 columns. The HILIC column temperature was 35℃; flow rate 0.3 mL / min; injection volume 2 μL; mobile phase composition A: 90% water + 2 mM ammonium formate + 10% acetonitrile, B: methanol + 0.4% formic acid. The C18 column temperature was 40℃; flow rate 0.4 mL / min; injection volume 2 μL; mobile phase composition A: water + 5 mM ammonium acetate + 0.2% ammonia, B: 99.5% acetonitrile + 0.5% ammonia. Throughout the analysis, samples were placed in an autosampler at 4℃, and QC samples were inserted into the sample queue to monitor and evaluate the system's stability and the reliability of the experimental data.
[0030] 3) Mass spectrometry analysis: Mass spectrometry analysis was performed using an AB 6500 + QTRAP mass spectrometer (AB SCIEX). ESI source conditions were as follows: Source temperature: 580℃, Ion Source Gas1 (GS1): 45℃, Ion Source Gas2 (GS2): 60℃, Curtain Gas (CUR): 35℃, IonSpray Voltage (IS): +4500 V or -4500 V in positive or negative modes, respectively, monitored in MRM mode.
[0031] 4) Data Analysis Workflow: Analyst software is used to extract peaks from the raw MRM data, obtaining the ratio of peak area to internal standard peak area for each substance. The content is then calculated based on the standard curve. Data analysis includes univariate statistical analysis, multidimensional statistical analysis, screening of differentially expressed metabolites, correlation analysis of differentially expressed metabolites, and KEGG pathway analysis.
[0032] (3) Results and Analysis Transcriptome and metabolome sequencing results are as follows Figure 1 As shown, where Figure 1 (a) is a scatter plot of differentially expressed genes. Figure 1 (b) is a bar chart of differential metabolites. Figure 1 (c) shows the KEGG enrichment map of the combined analysis of differentially expressed genes and metabolites. The results showed that the number of genes significantly upregulated and downregulated after arginine addition was 270 and 470, respectively, while the number of upregulated and downregulated metabolites was 38 and 21, respectively. KEGG enrichment analysis revealed that the levels of genes and metabolites involved in aminoacyl-tRNA synthesis, pyrimidine metabolism, the mTORC1 pathway, and the glycogen signaling pathway were significantly enriched in KEGG after arginine addition.
[0033] 3. Arginine activates the mTORC1 pathway in largemouth bass. To verify the regulatory effect of arginine treatment on the mTORC1 pathway in largemouth bass, liver samples were collected after an 8-week culture period for Western blot analysis. The specific experimental steps are as follows: (1) Western blot analysis 1) Protein sample preparation: Weigh 150 mg of frozen liver tissue into a glass homogenizer, add 1.5 mL of RIPA composite lysis buffer, homogenize on ice until no obvious tissue blocks are visible, then transfer to a new centrifuge tube and mix on a four-dimensional mixer for 30 min to achieve complete protein lysis. Then centrifuge at 4 ℃, 12000 r / min for 20 min, and place the supernatant in an ice-water bath. Protein concentration was then determined using a BCA protein assay kit, and the protein content of both groups was adjusted to be consistent before aliquoting and storing.
[0034] 2) SDS-PAGE electrophoresis: Add the sample protein extract to 5× protein loading buffer (reducing) at a ratio of 1:4, mix slightly and heat to boiling for 10 min; then add 20 µL to each well of the SDS-PAGE gel (with 5 µL of pre-stained standard protein marker added to the first well); finally, perform vertical electrophoresis on the gel plate under constant voltage (80V) for 120 min, until the bromophenol blue label reaches the bottom of the gel plate.
[0035] 3) Transfer to PVDF membrane: Place the gel after electrophoresis in the transfer solution for 1 min to remove the SDS bound on the membrane. At the same time, cut a PVDF membrane of appropriate size and soak it in methanol for 15 s to activate the PVDF membrane. Then, immerse the membrane and filter paper in the transfer solution for 3 min and arrange them from bottom to top in the order of filter paper-PVDF membrane-gel-filter paper. Cover the instrument and transfer the membrane for 50 min under constant current (0.4 mA / membrane).
[0036] 4) Blocking: After the PVDF membrane is transferred, wash it with PBST for 5 min, then block it in 5% skim milk powder solution for 2 h, and rinse it slightly with PBS solution after the transfer.
[0037] 5) Incubation with primary antibody: Dilute the primary antibody (S6, P-S6, 4EBP1, P-4EB-P1, β-ACTIN) 1:1000 in PBST buffer according to the instructions, then add it to the PVDF membrane with the protein sample, incubate at room temperature for 1 h, and wash 4 times with PBST for 5 min each time.
[0038] 5) Second antibody incubation: Add the prepared goat anti-rabbit second antibody, incubate at room temperature for 45 min, wash with PBST 3 times, 5 min each time; finally wash with PBS for 5 min.
[0039] 6) ECL development: Add ECL development solution and use a chemiluminescence imaging system for imaging. Adjust the parameters until the bands are clear.
[0040] (2) Results Analysis Western blot results as follows Figure 2 As shown in the figure. The results showed that the relative phosphorylation levels of S6 and 4EBP1 proteins in the mTORC1 pathway were significantly increased after the addition of arginine, indicating that arginine significantly activated the mTORC1 pathway of largemouth bass.
[0041] 4. Arginine promotes the secretion of IgM antibodies in the serum of largemouth bass. To verify the regulatory effect of arginine treatment on IgM antibody secretion in largemouth bass after Nocardia infection, serum samples were collected after infection, and the IgM antibody content in the serum was detected by Western blotting.
[0042] (1) Western blot analysis Referring to the above 3(1) method, the main difference is that the 5× protein loading buffer selected in the (2) SDS-PAGE electrophoresis step is replaced with Non-Reducing, and three standard sera are added to the first three wells respectively to calculate the absolute content of antibody IgM during loading.
[0043] (2) Results Analysis The results of IgM content in the serum of largemouth bass are as follows: Figure 3 As shown in the figure. The results showed that the serum IgM levels in both feed groups were between 5000-6000 μg / ml before Nocardia amberjack infection, with no significant difference between the two groups. However, after Nocardia amberjack infection, the serum IgM levels in the arginine-added group were significantly higher than those in the control group. This indicates that arginine significantly promotes the synthesis and secretion of more IgM antibodies into the serum by B cells through the aforementioned activation of the mTORC1 signaling pathway, thereby enabling them to perform neutralization and phagocytic functions against invading bacteria.
[0044] 5. Arginine inhibits cell death mediated by excessive activation of the NLRP3 inflammasome in largemouth bass after Nocardia amber infection. To investigate the regulatory effects of arginine treatment on NLRP3 inflammasome and cell death in largemouth bass infected with Nocardia amberjack, TUNEL assay was used to detect cell death in the liver of largemouth bass, and Western blotting was used to detect the expression of NLRP3 inflammasome-related proteins. The specific experimental steps are as follows: (1) TUNEL assay for cell death 1) Preparation of paraffin slices: a) Tissue fixation: Largemouth bass were anesthetized with MS-222, and liver tissue was taken and placed in 4% paraformaldehyde for more than 24 hours, ensuring that the fixative fully immersed the tissue.
[0045] b) Gradient dehydration and clearing: The fixed tissue was sequentially transferred to 60% anhydrous ethanol for 4 h, 70% anhydrous ethanol overnight (8-10 h), 80% anhydrous ethanol for 2 h, 90% anhydrous ethanol for 2 h, 95% anhydrous ethanol for 1.5 h (repeated twice), 100% anhydrous ethanol for 0.5 h (repeated twice); benzene alcohol solution for 10 min, xylene for 7 min (repeated twice).
[0046] c) Paraffin impregnation and embedding: Impregnate with paraffin at 60°C for 1 hour (repeat 3 times), then place the tissue in the center of the metal embedding frame and wait for the paraffin block to solidify.
[0047] d) Slicing: Trim the cut surface of the wax block and fix it on the microtome for slicing (thickness 5 µm). Transfer the slice to a spreader with water temperature of 41℃. After the cut surface is flat, take out the slice and put it in a constant temperature oven at 65℃ for 2 hours for later use.
[0048] 2) TUNEL staining: a) Dewax the slide in xylene for 5-10 min. Replace with fresh xylene and dewax again for 5-10 min. Add anhydrous ethanol for 5 min. Add 90% ethanol for 2 min. Add 70% ethanol for 2 min, then add distilled water for 2 min. b) Use a tissue biopsy pen to draw circles around the tissue, paying attention to the time to prevent the tissue from drying out; c) Dilute proteinase K (20 mg / ml) 1000 times with Tris-HCl, incubate at 37°C for 20 min, and wash 3 times with PBS for 5 min each time.
[0049] d) Add TUNEL detection solution (5 μL TdT enzyme + 45 μL fluorescent labeling solution per sample, freshly prepared) to the slide and incubate at 37°C in the dark for 1 h.
[0050] e) Wash 3 times with PBS, 5 min each time, and stain the cell nuclei with DAPI at room temperature for 8 min; f) After washing twice with PBS, absorb the water around the tissue with absorbent paper, add anti-fluorescence attenuation mounting medium, and then mount with a coverslip; use an Olympus BX53 fluorescence microscope to acquire fluorescence signals, and use the corresponding imaging system to analyze and process the images.
[0051] (2) Western blot detection The method is the same as 3(1).
[0052] (3) Results Analysis Western blot results of cell death and NLRP3 inflammasome-related protein in the liver of largemouth bass are as follows: Figure 4 (a) and Figure 4 As shown in (b). Figure 4 (a) The results showed that before Nocardia amberjack infection, cell death in the liver of largemouth bass in the arginine-added group was significantly lower than that in the control group; Nocardia amberjack infection led to a significant increase in the cell death rate in the liver of largemouth bass, but the arginine-added group was still significantly lower than that in the control group. This indicates that arginine inhibits cell death in the liver of largemouth bass. Figure 4 (b) The results showed that before Nocardia amberjack infection, the expression levels of ASC and Caspase-1 proteins in the liver of largemouth bass in the arginine-added group were significantly lower than those in the control group; while after Nocardia amberjack infection, the expression levels of NLRP3 and Caspase-1 proteins in the liver of largemouth bass in the arginine-added group were significantly lower than those in the control group, indicating that arginine inhibited the excessive activation of the NLRP3 inflammasome. Therefore, arginine inhibits cell death mediated by the excessive activation of the NLRP3 inflammasome in largemouth bass after Nocardia amberjack infection.
[0053] 6. Arginine protects the liver and other tissue structures of largemouth bass after Nocardia infection. To investigate the regulatory effect of arginine treatment on the liver and other tissue structures of largemouth bass infected with Nocardia amberjack, paraffin sections of the liver and other tissues were prepared and subsequently stained with Hematoxylin and Escherichia coli (H&E) for observation. The specific experimental procedures are as follows: (1) Preparation of paraffin slices of liver, head kidney and kidney of largemouth bass Paraffin sections were prepared as described in 5(1) above, with a thickness of 5 μm. The tissue was then retrieved using an adhesive glass slide and then 65°C. 0 Dry in a C oven for more than 2 hours.
[0054] (2) Hematoxylin-eosin (H&E) staining procedure 1) Dewaxing: Remove the sections from the oven and then treat them with xylene for 15 min (repeat three times); then treat them with anhydrous ethanol for 5 min (repeat twice), 75% anhydrous ethanol for 5 min, and wash with water for 3 min to complete the dewaxing process.
[0055] 2) Hematoxylin staining and differentiation: After treatment with hematoxylin staining solution for 3 min, wash twice with water, then soak in water for 3 min. After that, immerse the slide in differentiation solution (99 mL 70% alcohol + 1 mL concentrated HCl) for 3-5 s, rinse in water for 5 s, wipe dry and examine under a microscope.
[0056] 3) Blue return and eosin staining: After rinsing, immerse in blue return solution (99 mL 70% alcohol + 1 mL concentrated ammonia) for 3-5 seconds, remove and rinse under distilled water, then immerse in distilled water for 3 minutes; then place in 85% ethanol for 5 minutes, 95% ethanol for 5 minutes, and eosin solution (1 g of water-soluble eosin dissolved in 100 mL 85% alcohol) for 2-3 minutes.
[0057] 4) Gradient alcohol dehydration: anhydrous ethanol I 5 min, anhydrous ethanol II 5 min, anhydrous ethanol III 5 min, n-butanol 5 min, xylene I 5 min, xylene II 5 min.
[0058] 5) Mounting and imaging with neutral resin: Images were acquired in the microscope (Olympus) using AXIOVISION software.
[0059] (3) Results Analysis The histological results of the liver, head kidney, and body kidney of largemouth bass before and after Nocardia amberjack infection are as follows: Figure 5The results showed that before Nocardia amberjack infection, the liver cells of largemouth bass were cuboidal with round nuclei, arranged in a cord-like pattern, and supported by reticular fibers and connective tissue, with no significant difference between the two treatment groups. In the head kidney of largemouth bass, the radially arranged lymphoid cord structures, interspersed with blood sinuses filled with erythrocytes, were mainly composed of lymphocyte and granulocyte aggregation areas, interspersed with melanocyte-macrophages (MM) and a small number of MMCs, with no significant difference between the two treatment groups. The body kidney of largemouth bass mainly consisted of nephrons and collecting ducts. Nephrons included renal corpuscles and renal tubules. The pseudolymphoid tissue mainly consisted of lymphocytes, granulocytes, hemocytes, and melanocyte-macrophages. The renal corpuscle consisted of glomeruli and Bowman's capsule. The renal tubules were further divided into cervical segments, proximal convoluted tubules, and distal convoluted tubules, with no significant difference between the two treatment groups. Following Nocardia amberjack infection, liver cells gradually become disordered, with inflammatory cells scattered throughout, leading to the formation of hepatic granulomas. Some cells exhibit vacuolar degeneration accompanied by extensive cell membrane rupture. Obvious granulomatous nodules with extensive inflammatory cell infiltration are detected in the head kidney tissue. Granulomatous nodules appear in the parenchyma of the body kidney, with necrosis of the renal tubular epithelium and proliferation of connective tissue replacing nephrons. In the arginine-added group, no vacuolar degeneration was observed in the liver of largemouth bass after Nocardia amberjack infection, and cell membrane rupture was improved. Granulomatous nodules in the head kidney were significantly smaller, and the structural integrity of the renal tubular epithelium in the body kidney was significantly better. These results indicate that arginine supplementation improves the structural integrity of the liver, head kidney, and body kidney of largemouth bass after Nocardia amberjack infection, thus promoting tissue health and physical barrier function.
[0060] 7. Arginine promotes the survival rate of largemouth bass infected with Nocardia amberjack. To investigate the regulatory effect of arginine on the survival of largemouth bass after Nocardia amberjack infection, the survival rate of largemouth bass was counted after Nocardia amberjack challenge infection experiment.
[0061] The results are as follows Figure 6 As shown, the survival rate of largemouth bass after Nocardia amberjack infection in the control group was 43.75%, while the survival rate after Nocardia amberjack infection with arginine was 53.13%. These results indicate that the addition of arginine improved the survival rate of largemouth bass after Nocardia amberjack infection.
Claims
1. Application of arginine in promoting resistance of largemouth bass to Nocardia infection.
2. The application according to claim 1, characterized in that, The indicators of the largemouth bass's resistance to Nocardia amberjack infection include: high-throughput omics analysis, mTORC1 pathway activity, antibody content, cell death, tissue structure, and survival rate.
3. The application according to claim 2, characterized in that, The arginine activated the mTORC1 pathway in largemouth bass.
4. The application according to claim 3, characterized in that, The combined analysis of transcriptome sequencing and metabolome sequencing showed that after arginine addition, the levels of related genes and metabolites such as mTORC1 signaling pathway, aminoacyl-tRNA synthesis, pyrimidine metabolism, and glycogen signaling pathway were significantly enriched in KEGG. Further Western blot analysis detected a significant increase in the phosphorylation levels of S6 and 4EBP1 in the mTORC1 pathway, indicating that arginine significantly activated the largemouth bass mTORC1 pathway.
5. The application according to claim 2, characterized in that, The arginine promoted the secretion level of IgM antibodies in the serum of largemouth bass after Nocardia infection.
6. The application according to claim 5, characterized in that, The IgM antibody content in the serum of largemouth bass before Nocardia amberjack infection was 5000-6000 µg / mL, while the IgM antibody content in the serum of largemouth bass after Nocardia amberjack infection increased significantly after the addition of arginine, reaching 14000 µg / mL.
7. The application according to claim 2, characterized in that, The arginine acid inhibited cell death mediated by excessive activation of the NLRP3 inflammasome.
8. The application according to claim 7, characterized in that, The liver tissue of the largemouth bass showed significantly increased cell death and NLRP3 inflammasome protein expression after Nocardia amberjack infection. The addition of arginine can inhibit the cell death response mediated by excessive activation of NLRP3 inflammasome in largemouth bass.
9. The application according to claim 2, characterized in that, The arginine improved the tissue structure of the liver, head kidney, and body kidney of largemouth bass infected with Nocardia amberjack and increased the survival rate of largemouth bass infected with Nocardia amberjack.
10. The application according to claim 9, characterized in that, The liver, head kidney, and body kidney tissues of the largemouth bass showed signs of cell vacuolar degeneration and cell membrane rupture, granulomatous nodules, epithelial necrosis, and connective tissue hyperplasia after Nocardia amberjack infection. After the addition of arginine, the cell membrane rupture in the liver was slowed down, the granulomatous nodules in the head kidney were significantly smaller, and the integrity of the renal tubular epithelial structure in the body kidney was significantly better. At the same time, the survival rate of largemouth bass in the arginine-added group after Nocardia amberjack infection was significantly higher than that in the control group.
11. A functional feed additive that promotes resistance of largemouth bass to Nocardia amberjack infection, characterized in that, The functional feed additive includes arginine.