| Chinese mitten crab(Eriocheir sinensis),commonly known as river crab,also known as hairy crab,is loved by consumers because of its excellent flavor and nutrition.However,during the breeding process,the dissolved oxygen in the pond changes drastically day and night and the water environment is not properly controlled,which makes the crabs stay in a low oxygen state for a long time,which affects the growth and development and immunity of the crabs,resulting in poor body color,small size,and reduced physical fitness,which are prone to secondary infections.The diseased microorganisms may even die,which ultimately leads to a low catch rate and poor breeding efficiency.Dissolved oxygen is an important environmental factor in the breeding environment of river crabs,which directly affects the survival,growth,metabolism,digestion and immunity of river crabs.In this paper,biochemical and molecular biology methods were used to monitor the changes in dissolved oxygen in crab ponds,as well as the oxygen consumption rate and suffocation points of crabs.The effects of hypoxia on the physiology and biochemistry of crabs and the ultrastructure of mitochondria in different tissues were studied.-Transcriptomics analysis of crabs under reoxygenation conditions,screening of hypoxia-inducible factors,to provide a reference for further revealing the molecular mechanism of crabs responding to hypoxia stress.The main findings are as follows:1.Oxygen consumption is an important indicator of crustacean respiration metabolism,and suffocation point is an indicator of crustacean tolerance to hypoxia.This study measured the changes in pond dissolution,and the results showed that the dissolved oxygen in ponds at night was less than 4 mg/L for a long time.The minimum can be reduced to 1.6 mg/L(6:00 in the morning),that is,in the hypoxic phase;the oxygen consumption rate and suffocation point of different parts of the crabs were measured by the flowing water breathing chamber method and the static breathing chamber method.The results showed that the oxygen consumption rate and suffocation point of river crabs were negatively correlated with body weight.The oxygen consumption rate and suffocation point of large-sized river crabs were significantly lower than the oxygen consumption rate and suffocation point of small-sized river crabs.The average oxygen consumption rate of crabs at night was significantly higher than the average oxygen consumption during the day.2.In order to determine the effect of hypoxia stress on the mitochondrial ultrastructure and physiology and biochemistry of mitochondria in river crabs,this study performed hypoxia-reoxygenation treatments to determine the activities of SOD,T-ATPase,ALP,ACP and the contents of MDA and LDH in the crabs.The results showed that after 1 h of hypoxia treatment,the activities of SOD and ACP in the crabs were significantly reduced,and the contents of MDA and LDH,as well as the activities of T-ATPase and ALP were significantly increased.After 6 hours of hypoxia treatment,MDA content continued to increase,LDH content and T-ATPase and ALP activities decreased.SOD and ACP activities were equivalent to that of hypoxia treatment for 1 hour;SOD activity first increased and then decreased after reoxygenation,MDA,The content of LDH and the activity of ALP increased after decreasing,and the activity of T-ATPase and ACP increased during the reoxygenation phase.The results show that both hypoxia stress and reoxygenation recovery will have an impact on the physiological and biochemical indicators of crabs,so that they can cause stress to hypoxia-reoxygenation through regulating means such as their own anti-oxidative stress system and optimizing energy supply metabolism.Microscopic observation of mitochondrial tissue showed that after 6 hours of hypoxia stress,the number of mitochondria in the muscle tissue of crabs decreased,the inner cristae were blurred and arranged disorderly,some of the ridges disappeared,and the matrix was thin.After 12 hours of reoxygenation,the damage of mitochondria increased and vacuolization appeared.After 6 h of hypoxia stress,the cristae within the mitochondria of the crab gill tissue were destroyed,and the outer membrane was distorted and deformed after 12 h of reoxygenation.Part of the cristae disappeared,myeloid deformed,and some mitochondria were left with only microbubbles or residual membranes.The results show that hypoxia can cause severe damage to the mitochondria of river crabs,which cannot be recovered after 12 hours of reoxygenation,and the damage is even further aggravated.3.In order to reveal the molecular mechanism of the response of crabs under hypoxia-reoxygenation and screen the differentially expressed genes,this study analyzed the differential expression of certain genes of crabs under hypoxia-reoxygenation through high-throughput sequencing,and obtained 49.19 Gb of clean data and 27,233 new transcripts with functional annotations were assembled.Among them,26,819 sequences are homologous to genes in the Nr protein database;16,299 sequences are annotated to the KEGG database and classified into 284 metabolic pathways.In the hypoxic phase,103 and 251 differentially expressed genes were detected after exposure to severe hypoxia for 1 h and 6 h,respectively.In the reoxygenation phase,462 and 673 differentially expressed genes were identified during the 1 h and 12 h reoxygenation treatments,respectively.Hypoxic stress has a significant effect on 57 pathways in the 6-hour hypoxia group.Among them,the most significant ways to enrich the first three genes are "PPAR signaling pathway","Gap junction" and"Phototransduction-fly".In the reoxygenation phase,the most significant ways of enriching the first three genes are "ECM-receptor interaction","Lysosome" and"Phagosome".Seventeen differential genes were selected for fluorescence quantitative verification,and the results showed that the qRT-PCR results were consistent with the sequencing results up and down,which verified the accuracy of transcriptome sequencing.4.According to the results of full-length transcriptome sequencing,the key genes Hif-1α and Hif-1β in the hypoxic response of river crabs were screened and identified.The cDNAs were 5,377 bp and 2,868 bp in length,respectively,and the three ORFs coded for 1,057 and 551 amino acid residues,respectively.Base;both genes contain one HLH domain and two PAS domains.The analysis of the proteins that can interact with the Hif-1 gene of the river crab revealed that a total of 476 proteins(including 71 DEGs)may interact with the Hif-1 of the river crab in a direct or indirect manner.The GO enrichment analysis showed that,the vast majority of pathways are related to cellular processes,such as "oxidoreductase activity","transferase activity" and "small molecule binding".It shows that hypoxia stress activates a series of physiological activities of the crab body to adapt to hypoxia.In addition,the differences in Hif-1α and Hif-1β gene expression and tissue distribution of Eriocheir sinensis in ponds with well-grown aquatic plants and ponds without aquatic plants were studied.The results show that Hif-1α and Hif-1β are expressed in stomach,liver,intestine,gills,muscle,heart,eyestalk,and blood tissues,and their expression levels are higher in muscle,heart,and blood,and they are expressed in eyestalks.The least expression.Compared with the pond crab with aquatic grass,the expression of Hif-1α and Hif-1β in the pond crab with aquatic grass increased significantly,and the expression of Hif-1α in hepatopancreas,muscle and gills increased by 2.22,0.99,and 0.69 times,respectively.The expression of-1β in hepatopancreas,muscle and gill increased by 5.76,3.34,2.24 times,respectively.The results showed that an aquatic environment would have an impact on the organism of the crab,causing the crab to initiate a molecular regulation mechanism to respond to the hypoxic environment. |
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