The Apoptosis Responding Mechanism Under Air-Exposure Stress In Mud Crab Scylla Paramamosain

The mud crab(Scylla paramamosain Estampador,1949)is mainly dispersed in estuaries and coastal waters throughout the Indo-Pacific and Indian Ocean regions.It is also a commercially important marine species farmed in the Asian region especially in China South-East coastal areas.After captured,the crabs are transported inland for sell,which might take more than 72 h from south to north of China.To lower the transportation expenses,a dry approach has been adopted in practice.This method may cause aquatic animal functional hypoxia to gill and other internal tissues which could lead to a high morbidity and mortality and cause enormous economic losses.Furthermore,the air-exposure stress treatment of aquatic crustaceans may result in PO2,CO2 decrease and an internal hypoxia,which may cause oxidative stress and induce cell apoptosis.1.Antioxidant enzymes activities analysis and histological observation of mud crab under air-exposure and re-submersion stress.After air-exposure,the total structure of gill shrunken,gill lamellae collapsed and severe wrinkles were observed under SEM observation.Antioxidant enzyme analysis was performed to figure out antioxidant response in mud crab against air-exposure/re-submersion stress.The antioxidant enzymes activities were analyzed,including SOD,CAT,GST and T-AOC.In gill,after air-exposure stress SOD,CAT and GST activity significantly up-regulated at 24 h and 48 h and decreased to the normal level at 72 h and 96 h;after air-exposure followed by re-submersion stress,the activities rose significantly in 72 h and 96 h group but showed no significance in 24 h and 48 h group.In hepatopancreas,after air-exposure stress SOD activity significant up-regulated at 48 h,T-AOC showed high antioxidant capacity at 24 h and 48h,after air-exposure followed by re-submersion stress all the detected enzymes activity rose significantly at 72 h and 96 h.These results revealed that crab can up-regulate antioxidant activity against oxidative damage caused by air-exposure and re-submersion.2.Transcriptome analysis of mud crab gill under air-exposure stress.In this study,gill reference transcriptome,differentially expressed gene(DGE)analysis was conducted between air exposure 0 h,48 h and 96 h of mud crab Scylla paramamosain.Meanwhile,a total of 565,051 transcripts and 195,726 unigenes were assembled with a mean length of 724 bp and N50 length of 1,377 bp were observed.A total of 21,349 DEGs were identified between air exposure 48 h group,96 h group and control group,including 281 up-regulated genes and 21,068 down-regulated genes.Further Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis annotated 26,656 DEGs and some pathways were regarded as immune related,Apoptosis related and metabolism related pathways,such as IMD and Toll pathway,lysosome pathway,and apoptosis pathway.6 genes involved in multiple KEGG signaling pathways were validated by qRTPCR.This study demonstrates the first gill transcriptomic analysis challenged with air exposure stress in mud crab Scylla paramamosain and provides valuable gene resources for understanding the crab gill responding mechanism,which can provide new sight into the crab adaptation mechanism against air exposure stress.3.Molecular cloning and characterization of an inhibitor of apoptosis protein(IAP)from the mud crab under air exposure stressTo evaluate whether the inhibitor of apoptosis protein(IAP)was involved in apoptosis-resistance against air exposure stress in crustaceans,SpIAP was cloned and investigated for the first time.The full length of SpIAP was 3,351 bp,encoding a polypeptide of 662 amino acids,and the predicted SpIAP protein contained three BIR domains and one RING domain.BLASTP and phylogenetic analysis results showed that SpIAP was clustered together with other crustaceans IAPs.SpIAP was detected in all the examined tissues and predominantly expressed in hepatopancreas.When crabs were challenged with air exposure for 12 h,the expression level of SpIAP in hepatopancreas was significantly increased in experimental group compared with the control group.RNA interference assay and flow cytometry analysis exhibited that when SpIAP was silenced,the cell apoptotic rate significantly increased after 24 h air exposure treatment.These results suggested that SpIAP was involved in anti-apoptosis response induced by air exposure in mud crab S.paramamosain.4.BAX and Bcl-2 regulate apoptosis by activating mitochondrial pathway under air-exposure and re-submersion stress.To further analysis the apoptosis responding mechanism under air-exposure and re-submersion stress,the key activator/inhibitor of mitochondrial apoptosis pathway BAX/Bcl-2 were cloned and analyzed in mud crab.The homologue of the apoptosis regulator BAX was firstly identified in S.paramamosain and named as SpBAX.The coding region of SpBAX yielded a polypeptide of 278 amino acids,consisting of the defining motif of the BAXs family including Bcl-2 homology BH1-3 regions and a transmembrane(TM)domain.The homologue of B-cell lymphoma-2 was identified and named as SpBcl-2.The coding region of SpBcl-2 yielded a polypeptide of 207 amino acids,consisting of the defining motif of the Bcl-2 family including Bcl-2 homology BH1-4 regions and a transmembrane(TM)domain.The highest expression level of SpBAX and SpBcl-2 was detected in hepatopancreas.The expression level of SpBAX was up-regulated at 12 h in hepatopancreas and at 24 h in haemocytes after air exposure.Meanwhile,flow cytometry assay revealed that the over-expression of SpBAX in HEK293T cells could lead to cell apoptosis and SpBcl-2 could inhibit SpBAX induced apoptosis.Both SpBAX and SpBcl-2 were up-regulated under air-exposure and re-submersion stress.The SpBcl-2 showed higher expression level than SpBAX and result in the low ratio of BAX/Bcl-2 in air-exposure stress.However,during the re-submersion stress,SpBAX showed higher expression level than SpBcl-2 result in the up-regulation of BAX/Bcl-2 level and cause cell apoptosis.The TEM analysis further confirmed that the mitochondrial collapsed in the re-submersion group,indicating the activating of mitochondrial apoptosis pathway.These results indicated that the cloned SpBAX/SpBcl-2 might be involved in the response to air exposure and re-submersion stress by causing cell apoptosis.This study may be helpful to clarify the mechanisms of air-exposure stress response in mud crab.In conclusion,air-exposure stress may cause damage in crabs' gill and induce oxidative stress which further up-regulate the anti-oxidation capacity and activity.Meanwhile,anti-apoptosis protein SpIAP and SpBcl-2 were also up-regulated to inhibit the activity of SpBAX and Caspase to prevent the occurrence of apoptosis.Re-submersion stress may up-regulate the pro-apoptosis protein SpBAX and BAX/Bcl-2 ratio which might further destroy the mitochondrial structure and further activate mitochondrial apoptosis pathway.