Repeatable mild air exposure method for resisting acute stress of blue crabs, application and stress detection method

Abstract

The invention provides a repeated mild air exposure method for resisting acute stress of blue crabs, application and a stress detection method, and belongs to the technical field of aquaculture. According to the method, the blue crabs are subjected to repeated light air exposure treatment, so that the blue crabs are in a chronic stress state, the adaptability of the blue crabs to acute stress is improved, the stress resistance of the blue crabs is enhanced, the lethal problems of abnormal shelling, physiological disorder and the like caused by stress in the transportation process are greatly reduced, and the living body transportation death rate is remarkably reduced; and the survival rate and the commodity quality of the transported crabs are ensured. Economic loss can be directly reduced by reducing transportation loss of the blue crabs, resource waste is reduced, and efficient operation of a blue crab breeding industry chain is promoted; the stable supply of the high-quality live crabs can enhance the market competitiveness, promote the economic benefits of the whole chain of cultivation, transportation and sales, and provide support for the large-scale and intensive development of the industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aquaculture, and particularly relates to a repetitive mild air exposure method for resisting acute stress of blue crab, application and stress detection method. BACKGROUND

[0002] Aratus pisonii is the most common cultured species due to its fast growth, large size, high meat yield, delicious taste and adaptability to various culture systems. High-density culture, shortening of production cycle and other operations for maximizing profit and resource utilization rate may cause Aratus pisonii to be in a state of stress for a long time and prone to diseases. Therefore, it is necessary to use multidisciplinary methods (such as genetics, nutrition, technology and management) at various stages of the production cycle to reduce stress and reduce the scale and frequency of disease outbreaks.

[0003] For crabs such as Aratus pisonii, culture operation stress (such as air exposure) is one of the most important stress factors. Air exposure usually occurs during capture or transportation and can affect the homeostatic balance of animals and even cause death. Air exposure stress can weaken the ability of hemocyanin of crustaceans to bind oxygen, thereby reducing their oxygen consumption rate and causing hypoxic stress. Current studies have explored the effects of air exposure on crabs from the aspects of classic physiological parameters and omics, but the exposure durations in these studies are quite different, and the results are contradictory. Air exposure can affect the immune system of crabs, including antioxidant defense, nutrient metabolism, energy production, ion balance and almost all physiological mechanisms. Studies have shown that benign stress and adverse stress (stress that has a negative impact on animals) can trigger completely different body responses in the neuroendocrine system, behavior and other physiological mechanisms. However, there is no research to verify this phenomenon in crustaceans.

[0004] In the past decade since the completion of genome sequencing, high-throughput omics technologies, such as transcriptomics, proteomics, metabolomics, microbiomics and genomics, have been widely applied in aquaculture research. Transcriptomics aims to study all RNA molecules (including mRNA, rRNA, tRNA and other non-coding RNA) produced in a single cell or cell population. Currently, two analysis methods are mainly used to identify relevant pathways and functional terms under different treatments. The most commonly used method is ORA analysis, in which researchers set a threshold to screen differentially expressed genes and then use bioinformatics methods to determine relevant pathways and functional terms. This method is effective when a sufficient number of differentially expressed genes (usually more than 15% of the total number of genes) are detected. The other method is GESA analysis, which can identify over-represented gene or protein categories in the entire dataset. This exploratory analysis method can reveal pathways and functional terms that show changing trends and enrichment in the entire dataset, and is particularly suitable for studies where group / phenotype differences are not significant. These two methods have become routine and powerful tools for in-depth study of phenotypic changes in crustaceans and fish in the field of molecular biology, but there is currently no study that simultaneously uses both methods for comparative analysis.

[0005] Therefore, it is crucial to study the effects of air exposure on the growth, survival, stress response and other physiological mechanisms of Scylla paramamosain during the breeding cycle to alleviate the acute stress response of Scylla paramamosain. SUMMARY

[0006] The present application aims to provide a repetitive mild air exposure method for resisting acute stress of Scylla paramamosain, application and stress detection method, which can improve the adaptability of Scylla paramamosain to acute air exposure, enhance the stress resistance of Scylla paramamosain and reduce the mortality rate.

[0007] To achieve the above-mentioned application purposes, the present application provides the following technical solutions: The present application provides a repetitive mild air exposure method for resisting acute stress of Scylla paramamosain, comprising the following steps: The air exposure treatment is performed on Scylla paramamosain every 3-5 days.

[0008] Preferably, the number of air exposure treatments is 10-20 times.

[0009] Preferably, the time of air exposure treatment is 100-200 minutes each time.

[0010] The present application also provides an application of the above-mentioned repetitive mild air exposure method for resisting acute stress of Scylla paramamosain in reducing the stress of Scylla paramamosain.

[0011] The present application also provides a Scylla paramamosain stress detection method, comprising the following steps: (1) Taking the gill, hepatopancreas and muscle tissue of the blue crab as genetic information, and performing sequencing analysis; (2) According to the sequencing results, whether the blue crab is stressed is judged.

[0012] Preferably, in step (2), the method for judging whether the blue crab is stressed comprises gene detection or pathway change detection.

[0013] Preferably, the genes to be detected in the gene detection include two or more of Y+L amino acid transporter 2, serine / threonine protein phosphatase 6 anchor repeat regulatory subunit C, heat shock protein 70, heat shock protein 90, DNA replication licensing factor MCM4-like protein, MCM7, ankyrin-1, D-beta-hydroxybutyric acid dehydrogenase, FreD protein, organic cation transporter, 2-hydroxyacyl sphingosine 1-beta-galactosyltransferase or glutamine synthetase.

[0014] Preferably, the pathway change includes two or more of the following: Up-regulation of DNA replication and endoplasmic reticulum protein processing pathways in gill and muscle tissues; Down-regulation of ribosome pathways in gill, hepatopancreas and muscle tissues; Down-regulation of mTOR signaling pathways in hepatopancreas; Down-regulation of amino acid metabolism pathways in hepatopancreas; Up-regulation of DNA repair pathways in gill, hepatopancreas and muscle tissues.

[0015] Compared with the prior art, the present application has the following beneficial effects: (1) The present application repeatedly exposes the blue crab to a mild air environment, so that the blue crab is in a chronic stress state, improves the adaptability of the blue crab to acute air exposure, enhances the stress resistance of the blue crab, greatly reduces the problems of abnormal molting, physiological disorder and other fatal problems caused by stress during transportation, significantly reduces the mortality rate of live transportation, and ensures the survival rate and commodity quality of the crabs after transportation. The reduction of transportation loss of the blue crab can directly reduce economic losses, reduce resource waste, and promote the efficient operation of the blue crab breeding industry chain; stable supply of high-quality live crabs can enhance market competitiveness and drive the economic benefits of the whole chain of breeding, transportation and sales to improve, providing support for the industrialization and intensification of the industry.

[0016] (2) The present application detects the influence of repeated mild air exposure on male and female crabs, reveals the molecular mechanism of the stress resistance adaptation of crabs induced by repeated mild air exposure, and provides key technical data for analyzing the physiological and biochemical basis of the air exposure adaptation of crustaceans. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below only relate to some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0018] FIG. 1 is the identification result of differentially expressed genes in each group in the embodiments of the present application, wherein A is the liver and pancreas tissue contrast volcano plot between male and female individuals; B is the liver and pancreas contrast volcano plot between the female control group and the female RMS group; C is the muscle tissue contrast volcano plot of the male and female RMS groups; Figure 2 FIG. 2 is the combined analysis result of GSEA and ORA of the gill, liver and pancreas and muscle tissues between male and female individuals in the embodiments of the present application; Figure 3 FIG. 3 is the combined analysis result of GSEA and ORA of the gill, liver and pancreas and muscle tissues between female individuals in the embodiments of the present application; Figure 4 FIG. 4 is the combined analysis result of GSEA and ORA of the gill, liver and pancreas and muscle tissues of the female RMS group in the embodiments of the present application; Figure 5 FIG. 5 is the combined analysis result of GSEA and ORA of the gill, liver and pancreas and muscle tissues of the male individual in the embodiments of the present application; Figure 6 FIG. 6 is the combined analysis result of GSEA and ORA of the gill, liver and pancreas and muscle tissues of the male RMS group in the embodiments of the present application; Figure 7 FIG. 7 is the combined analysis result of GSEA and ORA of the gill, liver and pancreas and muscle tissues of the combined male and female groups in the embodiments of the present application; Figure 8 FIG. 8 is the combined analysis result of GSEA and ORA of the gill, liver and pancreas and muscle tissues of the combined male and female RMS groups in the embodiments of the present application. DETAILED DESCRIPTION

[0019] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, features and embodiments of the present application.

[0020] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range and any other stated value or intermediate value within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example This invention provides a method for reducing acute stress in mud crabs through repeated mild air exposure, the specific steps of which are as follows: (1) Repeated mild air exposure treatment (1.1) Animal ethics All animal handling and experimental procedures were reviewed and approved by the Animal Ethics Committee of Shantou University and were implemented prudently in strict accordance with the ethical guidelines of the "Guidelines for the Care and Use of Laboratory Animals".

[0025] (1.2) Repeated mild air exposure treatment The mud crabs were purchased from a local crab farm in Niutianyang (located in Shantou City, Guangdong Province, China).

[0026] A three-dimensional crab farming system (crab apartments) was used. The rearing tanks measured 35 cm × 30 cm × 20 cm, each containing approximately 5.3 liters of water. The system was equipped with an indoor circulating water system, replenishing 10% seawater daily (salinity 21±2.4, water temperature 25±2.4℃). During the experiment, water quality parameters were monitored and maintained at optimal levels: dissolved oxygen concentration (>6.0 mg / L), pH value (7.0-8.0), and total ammonia nitrogen concentration (<0.2 mg / L) all met the crab growth requirements. Clams were fed to satiety every evening, and uneaten food and molted shells (primarily molting at night) were removed the following morning.

[0027] Four treatment groups were set up: female control group, male control group, female RMS group and male RMS group, with 6 crabs (initial body weight 26.9 ± 4.3 g) in each group, a total of 24 crabs.

[0028] The RMS group was exposed to air for 150 minutes every four days, with a total of 15 exposure treatments over 63 days. At the end of the experiment, all remaining crabs were fed in the evening, and acute air exposure treatment was started the next morning. After 24 hours of continuous air exposure, sampling was carried out, and the experimental crabs were fasted for 24 hours before sampling. Liver, gills and muscle tissues were collected for transcriptome analysis and gene expression detection. To ensure that the samples from each group were collected about 24 hours after the start of air exposure stress, all sampling was carried out during the day. The acute air exposure treatment continued until all experimental crabs died, and the number of deaths was recorded every 6 hours during this period. The collected samples were immediately frozen with liquid nitrogen and stored in a -80°C ultra-low temperature freezer for subsequent experimental analysis. To maintain consistency between data, different tissues from the same crab were used for inter-group comparison.

[0029] (2) Analysis of growth performance The growth performance indicators of crabs were determined according to the following formula, including final body weight, weight gain, specific growth rate (%), increase in carapace width (mm), increase in carapace length (mm), feed conversion rate, molting rate (%) and survival rate (%). The results are shown in Table 1.

[0030] Weight gain = final body weight - initial body weight Specific growth rate (SGR, % / day) = 100 x (ln (final body weight) - ln (initial body weight)) / experimental days Carapace width / length increase = final carapace width / length - initial carapace width / length Feed conversion rate = total feed intake / total weight gain (g) Molting rate (%) = (total number of molts / total number of experimental crabs) x 100 Survival rate (%) = (number of surviving crabs at the end / number of initial stocked crabs) x 100 Table 1 Growth performance of Trachinotus ovatus after 63 days of repetitive mild air exposure treatment

[0031] Table 1 shows that no significant differences were found among the groups of mud crabs in terms of weight gain, specific growth rate, carapace width increase, carapace length increase, feed conversion ratio, molting rate, and survival rate. Although not statistically significant, the weight gain of the repeated mild stress group was better after 63 days of culture (67 g vs 62 g). The acute air exposure experiment showed that the survival time of the repeated mild stress group (143.6 h) was significantly longer than that of the control group (127.71 h), and the effect of repeated mild stress was confirmed to be statistically significant by two-way ANOVA.

[0032] At the behavioral level, clear signs of adaptive response were also observed: In the final week of the experiment, crabs in the mildly stressed group exhibited lower reactivity and aggressive behavior when captured. Specifically, capture in the mildly stressed group took 20-30 seconds in the early stages, but only 5-10 seconds in the later stages. Simultaneously, during air exposure, the mildly stressed group also showed a calmer state within the rearing tank.

[0033] (3) Transcriptome analysis (3.1) RNA extraction and cDNA synthesis Total RNA was extracted from three tissues (gills, muscle, and hepatopancreas) of individual mud crabs (Scylla serrata) using a commercially available RNA extraction kit. Each tissue was taken from different individuals to ensure biological independence (a total of 12 females and 12 males). Immediately after dissection, the tissues were immersed in RNA preservation tissue stabilization solution (Nanjing Novozymes, China) and stored at -80°C until further processing. RNA was isolated using AG RNAex Pro RNA extraction reagent (Hunan Kangrun Biotechnology, China) according to the product instructions.

[0034] RNA integrity was confirmed by 1% agarose gel electrophoresis. Nucleic acid concentration and purity were determined using a Thermo Scientific NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA). RNA samples meeting purity requirements (230 nm / 260 nm absorbance ratio greater than 1.8) were selected for subsequent analysis and stored at -80°C until further experiments were conducted.

[0035] Add 1 μg RNA, 5 μL HiScript IV All-in-one Ultra RT Super Mix for qPCR (Nanjing Novozymes, China), and RNase-free water to a 20 μL reaction volume. The reverse transcription reaction is carried out at 50°C for 10 minutes, followed by a 5-second incubation at 85°C to terminate the reaction. Dilute half of the synthesized cDNA 20-fold, using 1 μL of the diluted product for each qPCR reaction.

[0036] The detection structure showed that the number of valid reads of all samples was between 32,443,640 and 43,621,116; the number of valid bases fluctuated in the range of 9,661,265,660 to 13,021,824,299; the GC content was distributed in the interval of 38.63% to 50.26%; and the proportion of bases with a quality value ≥ Q30 was 93.22%-96.14%. The SF2 file also showed that the total number of genome alignment reads of all samples was 64,887,280-87,242,232; the alignment success rate was 87.01%-95.99%; the unique alignment rate was between 78.36%-91.13%; and the multiple alignment rate ranged from 3.51% to 14.14%.

[0037] (3.2) Library construction and sequencing of transcriptome analysis 1 μg of RNA was taken from each sample as the starting material for library construction. The Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina (Qingdao Baimaikesheng Biotechnology Co., Ltd., China) was used to construct the sequencing library, and the operation strictly followed the manufacturer's recommendations.

[0038] mRNA was isolated from total RNA by magnetic beads with poly-T oligonucleotides. Then the first strand cDNA was synthesized, and the second strand cDNA was synthesized. The residual overhanging ends were converted to blunt ends by exonuclease / polymerase activity. After adenylation at the 3' end of the DNA fragments, NEBNext adaptors with hairpin loop structures were ligated for hybridization. The library fragments were purified using the AMPure XP system (Beckman Coulter, USA). 3 μL of USER enzyme (NEB, USA) was incubated with the cDNA after fragment selection and adaptor ligation at 37°C for 15 minutes, followed by 95°C treatment for 5 minutes, and finally PCR amplification. Phusion High-Fidelity DNA polymerase, universal PCR primers, and Index primers were used for PCR amplification. Finally, the PCR product was purified by the AMPure XP system, and the library quality was evaluated using the Agilent 2100 Bioanalyzer. The library was sequenced on the Illumina NovaSeq platform according to the manufacturer's operating specifications, generating 150 bp paired-end sequencing reads.

[0039] (3.3) Gene analysis (3.3.1) Identification of differentially expressed genes and gene set enrichment analysis Differentially expressed genes (DEGs) were identified by DESeq2 software (1.40.1) in R language (4.3.0) with the screening criteria of corrected p-value < 0.05 and fold change of expression ≥ 1.5. Hierarchical clustering analysis of differentially expressed genes was performed using the Pheatmap package (1.0.12) in R software (4.3.0). Gene set enrichment analysis (GSEA) was performed by clusterProfiler (4.8.1) in R language (version 4.3.0), with a corrected p-value of ≤ 0.05 as the significant threshold. Overrepresentation analysis and gene set enrichment analysis were performed using Kyoto Encyclopedia of Genes and Genomes (KEGG; http: / / www.genome.jp / kegg / ) and Gene Ontology (GO; http: / / geneontology.org) databases, respectively, and the results are shown in Figure 1.

[0040] The volcano plot on the right shows the distribution of up-regulated and down-regulated differentially expressed genes in each comparison group. After 63 days of experiment, the comparison of liver and pancreas tissues between male and female individuals had the most differentially expressed genes, with 385 up-regulated genes and 556 down-regulated genes detected (Figure 1, panel A). The comparison of liver and pancreas tissues between female control and female RMS groups detected only 161 up-regulated and 147 down-regulated genes, respectively (Figure 1, panel B). The comparison of muscle tissues between male and female RMS groups before and after acute air stress showed the most significant transcriptomic changes, with 558 up-regulated and 790 down-regulated genes (Figure 1, panel C). The muscle tissues of female individuals before and after acute air stress showed the least difference, with only 383 differentially expressed genes detected.

[0041] (3.3.1) Effects of repeated mild air exposure When integrating the male and female datasets and comparing the conventional rearing conditions with the RMS conditions, it was found that only the liver and pancreas data between the two groups showed significant differences.

[0042] There were 168 genes up-regulated and 152 genes down-regulated between the two groups. Protein processing in endoplasmic reticulum and apoptosis pathways were significantly enriched in the RMS group, while tyrosine metabolism, fatty acid metabolism, glycerophospholipid metabolism and lysozyme-related pathways were down-regulated. In the combined male and female RMS group, some GO terms were highly enriched, including oxidoreductase activity (MF:0016491), glycolytic process (BP:0006096), fatty acyl-CoA metabolic process (BP:0035337), coenzyme A-ligase activity (MF:0016405) and ATPase-coupled transmembrane transport activity (MF:0042626). In contrast, glycerophospholipid catabolic process (BP:0046475), branched-chain amino acid transport (BP:0015803), serine-type endopeptidase activity (MF:0004252), GO terms related to neuronal development / projection / recognition and extracellular region (MF:0005576) were all lower in the combined male and female RMS group than in the combined male and female control group (Supplementary File SF4). Combined GSEA and ORA analysis showed that the commonly changed pathways in the liver and pancreas tissue were the significant enrichment of apoptosis and endoplasmic reticulum protein processing pathways, and the down-regulation of tyrosine metabolism pathway Figure 2 .

[0043] (4) Comparison between the treatment groups before and after 24 hours of AAE stress After 24 hours of acute air exposure stress, gill, hepatopancreas and muscle tissue samples were collected from male and female crabs to test whether crabs reared under repeated mild stress conditions would exhibit the same stress response as the control group when faced with acute air exposure. Changes in the four experimental groups before and after air exposure were compared, and two additional comparisons of combined male and female datasets were performed.

[0044] (4.1) Female analysis (4.1.1) Female individual analysis GSEA analysis showed that acute air exposure stress inhibited ribosome, amino acid biosynthesis and tyrosine metabolism pathways in gill tissues, and also inhibited glycolysis, amino acid biosynthesis and oxidative phosphorylation pathways in muscle tissues. ORA analysis showed that amino acid biosynthesis pathway was down-regulated in gill, hepatopancreas and muscle tissues. Key pathways related to energy production and amino acid metabolism, such as arginine biosynthesis, tyrosine and tryptophan metabolism, proteasome, pyruvate metabolism and pentose phosphate pathway, were among the most significantly down-regulated pathways. On the contrary, endoplasmic reticulum protein processing and glycan biosynthesis pathways were enriched in gill and muscle tissues (GSEA analysis), and ORA analysis also confirmed the activation of endoplasmic reticulum protein processing pathway in these tissues. Acute air exposure stress also positively enriched some GO functional terms, including DNA replication initiation (BP:0006270), DNA helicase activity (MF:0003678), MCM complex (CC:0042555), protein folding (BP:0006457) and ATP binding (MF:0005524). The expression levels of functional terms such as oxidoreductase activity (MF:0016491), ubiquitin-protein ligase binding (MF:0031625), acyl-CoA metabolic process (BP:0006637), protein catabolic process (BP:0030163), threonine-type endopeptidase activity (MF:0004298) and proteasome core complex (CC:0005839) were reduced due to acute air exposure stress.

[0045] The common pathway changes in gill tissues showed by the two analysis methods included enrichment of DNA replication, endoplasmic reticulum protein processing, amino sugar and nucleotide sugar metabolism, and down-regulation of tyrosine metabolism and amino acid biosynthesis. In muscle tissues, the common pathway changes were enrichment of endoplasmic reticulum protein processing pathway, and down-regulation of glycolysis / gluconeogenesis, amino acid biosynthesis and purine metabolism pathways. Figure 3 )。

[0046] (4.1.2) Female RMS group The female RMS group did not show completely consistent results, in which the ribosome pathway was the main down-regulated pathway in gill and muscle tissues. In hepatopancreas, amino acid biosynthesis and dorsal-ventral axis formation pathways were down-regulated together with tyrosine metabolism, Hippo signaling pathway, MAPK signaling pathway, etc. On the other hand, similar to the female control group, the endoplasmic reticulum protein processing pathway in muscle tissues showed enrichment. Both analysis methods showed that DNA replication was the main enriched pathway in all tissues.

[0047] In gill tissue, several pathways related to cellular structure such as base excision repair, nucleotide excision repair, and apoptosis were also upregulated. Together, both approaches confirmed enrichment of base excision repair, nucleotide excision repair, and DNA replication pathways in gill tissue, while ribosome pathway expression was downregulated. In hepatopancreas, GSEA and ORA together showed upregulation of retinol metabolism pathway, and downregulation of dorsal-ventral axis formation and amino acid biosynthesis pathways. DNA replication and endoplasmic reticulum protein processing pathways were enriched in muscle tissue, while ribosome expression was downregulated Figure 4 Under AAE stress, the most significantly enriched pathways in the female RMS group included DNA replication initiation (BP:0006270), DNA helicase activity (MF:0003678), MCM complex (CC:0042555), carbohydrate derivative metabolic process (GO:1901135), carbohydrate catabolic process (BP:0016052), and UDP-glucosyltransferase activity (MF:0008194). Correspondingly, female gametogenesis (BP:0007292), transmembrane transporter activity (MF:0022857), ribosome (CC:0005840), regulation of nitrogen compound metabolic process (BP:0051171), protein binding (MF:0005515), nucleus (CC:0005634), translation (BP:0006412), and ribosomal structural constituent (MF:0003735) were expressed at lower levels in this group under AAE stress (SF4). Overall, the female RMS group had more differentially expressed genes than the female control group, with more than 700 differentially expressed genes observed in both gill and hepatopancreas tissue.

[0048] (4.2) Male analysis (4.2.1) Male individual analysis In male individuals, AAE stress led to downregulation of protein synthesis (ribosome, ribosome biogenesis) pathways in all three tissues. The proteasome pathway in gill and muscle tissues, and the oxidative phosphorylation pathway in gill tissue also showed downregulation. Protein processing in endoplasmic reticulum was one of the most significantly enriched pathways in hepatopancreas and muscle tissues, while DNA replication was the most significantly enriched pathway in gill and muscle tissues. GSEA and ORA analyses showed a decrease in the expression level of ribosome, oxidative phosphorylation, spliceosome, and proteasome pathways in gill tissue. In muscle tissue, there was a decrease in the degree of enrichment of DNA replication and ribosome pathways. AAE stress significantly enriched the following functional terms: signal transduction (BP:0007165), stress response (BP:0006950), ATP binding (MF:0005524), MCM complex (CC:0042555), mitochondrial translation (BP:0032543), oxidoreductase activity (MF:0016491), mitochondrion (CC:0005739), DNA replication initiation (BP:0006270), and DNA helicase activity (MF:0003678). In addition, translation (BP:0006412), ribosomal structural constituent (MF:0003735), and ribosome (CC:0005840) were inhibited by AAE stress in all three tissues of male individuals (SF4). The gill tissue had the most differentially expressed genes, with 647 genes upregulated and 701 genes downregulated Figure 5 .

[0049] (4.2.2) Male RMS group In the male RMS group after 24 h AAE stress, amino acid biosynthesis, oxidative phosphorylation and mTOR signaling were the three core pathways down-regulated in all three tissues. Ribosome pathway was also down-regulated in gill and muscle tissues. Endoplasmic reticulum protein processing was the most significantly enriched pathway in muscle tissue, while DNA replication was among the top enriched pathways in all tissues. Some lipid metabolism related pathways were up-regulated in all three tissues, including linoleic acid metabolism, steroid hormone biosynthesis, unsaturated fatty acid biosynthesis and arachidonic acid metabolism. The most significantly up-regulated GO terms in all tissues of this group under AAE stress were DNA replication initiation (BP:0006270), DNA helicase activity (MF:0003678) and MCM complex (CC:0042555). While the most significantly inhibited GO terms were ATP synthesis coupled electron transport (BP:0042773), oxidative phosphorylation (BP:0006119), NADH dehydrogenase activity (MF:0008137), translation (BP:0006412), ribosomal structural constituent (MF:0003735) and ribosome (CC:0005840) (SF4). The joint analysis of GSEA and ORA showed that the common changed pathways in gill tissue were up-regulation of DNA replication pathway and down-regulation of oxidative phosphorylation and amino acid biosynthesis; the common pathways in hepatopancreas tissue were enrichment of DNA replication and down-regulation of mTOR signaling pathway; both methods showed enrichment of DNA replication, endoplasmic reticulum protein processing and N-glycan biosynthesis pathways and down-regulation of ribosome pathway in muscle tissue Figure 6 .

[0050] (4.3) Analysis of the combined male and female groups Combined datasets of male and female individuals before and after 24 hours of AAE stress were examined to observe overall response patterns regardless of sex. Inhibition of protein synthesis (ribosome pathway) was clearly observed in all tissues. Furthermore, upregulation of DNA replication and endoplasmic reticulum protein processing was observed in gill and muscle tissues. Finally, glycerophospholipid metabolism and steroid biosynthesis were the most predominantly enriched pathways in the hepatopancreas. In the combined male and female group, AAE stress significantly enriched the following GO terms: DNA replication initiation (BP:0006270), DNA helicase activity (MF:0003678), MCM complex (CC:0042555), GO terms related to neuronal development / projection / recognition, rRNA processing (BP:0006364), RNA binding (MF:0003723), and ATP binding (MF:0005524). Translation (BP:0006412), ribosome structural composition (MF:0003735), and ribosomes (CC:0005840) were the most significantly suppressed GO terms (SF4) across all tissues. Combined GSEA and ORA analysis showed upregulation of DNA replication and endoplasmic reticulum protein processing pathways (enrichment) in both gill and muscle tissues, while downregulation of the ribosome pathway was observed in gill, hepatopancreas, and muscle tissues. Figure 7 ).

[0051] (4.4) Male-female combined RMS group Merging data from female and male RMS groups and analyzing their changes under AAE stress revealed a significant increase in the number of significantly altered genes, with approximately 1300 differentially expressed genes observed in gill and muscle tissues, respectively. These changes far exceeded those observed in the control group. The stress response in the RMS group was more complex and diverse: in addition to the aforementioned ribosome pathway inhibition, upregulation of DNA replication and endoplasmic reticulum protein processing pathways, downregulation of the mTOR signaling pathway and various amino acid metabolisms (tyrosine, alanine, aspartate, glutamic acid, arginine, cysteine, and methionine) was also observed. Simultaneously, upregulation of certain DNA repair pathways (such as excision repair and mismatch repair) was observed in all three tissues. Joint analysis by GSEA and ORA revealed common changes in gill tissue, including high expression of DNA replication, base excision repair, and mismatch repair, as well as downregulation of ribosome, amino sugar, and nucleotide sugar metabolism. In hepatopancreatic tissue, the common pathways were enrichment of DNA replication and decreased expression of the mTOR signaling pathway and amino acid biosynthesis. In muscle tissue, there was a common enrichment of DNA replication, N-glycan biosynthesis, and endoplasmic reticulum protein processing pathways, as well as downregulation of the ribosome pathway. Figure 8 ).

[0052] Similar to the combined group, DNA replication initiation (BP:0006270), DNA unwinding enzyme activity (MF:0003678), MCM complex (CC:0042555), and rRNA metabolic process (BP:0016072) were the most significantly enriched GO terms in all tissues of the combined RMS group. Accordingly, similar to the combined group, translation (BP:0006412), ribosomal structural constituent (MF:0003735), and ribosome (CC:0005840) were the most significantly inhibited GO terms in all tissues under AAE stress (SF4).

[0053] Overall, the number of differentially expressed genes in male individuals was much greater than that in female individuals, indicating that male individuals were more sensitive to AAE stress or at least exhibited more diverse stress responses, whether in the control group or the RMS group. In addition, the response of the RMS group to AAE stress was more complex and intense than that of the control group.

[0054] (5) Key differentially expressed genes as potential biomarkers First, differentially expressed genes with a fold change of more than 2 in all comparisons of the three tissues at the end of the experiment (ST5) and after 24 hours of AAE stress were selected. Subsequently, genes that appeared at least 4 times in the comparisons and whose functions had been reported and key differentially expressed genes that changed after 24 hours of AAE stress were screened from them. Overall, these genes can be used as good biomarkers of air exposure.

[0055] The results showed that nearly 30% of the genes belonged to the amino acid, carbohydrate, and lipid transport and metabolism categories. At the same time, these genes were also involved in inorganic ion transport and metabolism, energy generation and conversion, and many other functions. Y+L amino acid transporter 2, serine / threonine-protein phosphatase 6, ankyrin repeat regulatory subunit C, heat shock protein 70, heat shock protein 90, DNA replication licensing factor MCM4-like protein, MCM7, ankyrin-1, D-beta-hydroxybutyrate dehydrogenase, and other genes appeared at least 8 times in the comparison list. Heat shock protein 70, FreD protein, organic cation transporter, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase, and glutamine synthetase, and other genes appeared at least 12 times in the key gene comparison list, and these genes had the potential to be used as stress biomarkers.

[0056] The above only describes preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A method of repetitive mild air exposure to resist acute stress in blue crabs, comprising, The method comprises the following steps: The air exposure treatment is performed on the mud crab once every 3-5 days.

2. The method for repetitive mild air exposure to prevent acute stress in blue crabs of claim 1, wherein, The number of times of the air exposure treatment is 10-20 times.

3. The method for repetitive mild air exposure to prevent acute stress in blue crabs of claim 1, wherein, The time of the air exposure treatment is 100-200 minutes each time.

4. Application of the repeated mild air exposure method for resisting acute stress of mud crab according to any one of claims 1-3 in reducing acute stress of mud crab.

5. A method for detecting stress in mud crabs, characterized in that, The method comprises the following steps: (1) Take the gill, hepatopancreas and muscle tissue of the mud crab to obtain gene information and perform sequencing analysis; (2) Determine whether the mud crab is stressed according to the sequencing result.

6. The method according to claim 5, wherein the stress of the blue crab is detected. In step (2), the method for determining whether the mud crab is stressed comprises gene detection or pathway change detection.

7. The method according to claim 6, wherein the stress of the blue crab is detected by measuring the amount of the at least one of the stress biomarkers in the sample. The genes to be detected in the gene detection comprise two or more of Y+L amino acid transporter 2, serine / threonine protein phosphatase 6 anchor repeat regulatory subunit C, heat shock protein 70, heat shock protein 90, DNA replication licensing factor MCM4-like protein, MCM7, ankyrin-1, D-beta-hydroxybutyric acid dehydrogenase, FreD protein, organic cation transporter, 2-hydroxyacyl sphingosine 1-beta-galactosyltransferase and glutamine synthetase.

8. The method according to claim 7, wherein the stress of the blue crab is detected. The pathway change comprises two or more of the following: Up-regulation of DNA replication and endoplasmic reticulum protein processing pathways in the gill and muscle tissue; Down-regulation of ribosome pathways in the gill, hepatopancreas and muscle tissue; Down-regulation of mTOR signaling pathways in the hepatopancreas; Down-regulation of amino acid metabolism pathways in the hepatopancreas; Up-regulation of DNA repair pathways in the gill, hepatopancreas and muscle tissue.

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