A Preliminary Study On The Autophagy And Apoptosis Of Hemocytes In Hard Clam Mercenaria Mercenaria And The Conditions Of Transportation And Preservation Under Air Exposure

The hard clam(Mercenaria mercenaria),naturally inhabits on the west coast of the Atlantic Ocean of North America,ranging from the intertidal zone to subtidal zone.In 1997,academician Zhang Fusui et al.first introduced the hard shell clam from the United States to China,and it has become an important shellfish pond culture in China,with the cultivation area of hundreds of thousands of mu.Hard clams are often subjected to air exposure due to natural tides and during long distance transportation and preservation.Exposure to air can result in physiological stress on shellfish,including desiccation,hypoxia and starvation.Therefore,air exposure is considered as one of the key stress factors influencing the survival of shellfish.The hard clams exhibit remarkable tolerance ability to air exposure and can survive for at least 10 days under air exposure at 15℃.Consequently,hard clam can serve as a suitable model to explore the molecular tolerance mechanism of intertidal shellfish to air exposure.In this study,the process of autophagy and apoptosis of hemocytes in hard clams exposed to air were investigated.Firstly,the sub-populations of hemocytes and the change of their number ratio under air exposure were identified.Additionally,the reactive oxygen species(ROS)level and the activities of superoxide dismutase(SOD)and catalase(CAT)were measured to investigate the oxidative stress response of hemocytes in hard clams exposed to air.At the cellular level,the level of autophagy and apoptosis of hemocytes in hard clams exposed to air were investigated through transmission electron microscopy and flow cytometry.At the molecular level,the transcriptome sequencing was performed on the hemocytes of hard clams under different durations of air exposure and the weighted correlation network analysis(WGCNA)was used to reveal the dynamic molecular response of hemocytes in hard clams exposed to air.This study also provided new insights on the molecular tolerance mechanism to air exposure in hard clams from the perspectives of autophagy and apoptosis of hemocytes.1.Identification of the sub-population of hemocytes in hard clams and oxidative stress response under air exposureIn this study,it was determined that the hemocytes of hard clams were composed of granulocytes and hyalinocyte,and the number ratio was about 6:4.The proportion of granulocytes in the total hemocytes decreased,while the proportion of hyalinocyte in the total hemocytes increased during air exposure.During air exposure,the glucose concentration in the hemocytes of hard clams decreased continuously,indicating the state of starvation.At the early stage of air exposure,ROS significantly accumulated in the hemocytes,resulting in oxidative stress.In this case,SOD activity was up-regulated to remove ROS.However,when the ROS accumulation reaches high level,the antioxidant effect of SOD was lost.2.Autophagy and apoptosis of hematocyte in hard clams under air exposureAt the cellular level,this study found that the autophagy process of hemocytes was activated on the first day after air exposure detected by transmission electron microscopy,confocal laser scanning microscope and flow cytometry.The autophagy level increased first and then decreased during the whole process of air exposure.This result suggested that autophagy was activated to remove damaged intracellular proteins and organelles under the induction of starvation and ROS accumulation.On the 20 th day of air exposure,the autophagy level of hemocytes decreased significantly,whereas apoptosis rate increased significantly.This results indicated that the up-regulation of autophagy level was limited.With the increase of stress intensity,hard clams could accelerate the death of severely damaged cells by activating apoptosis,with the aim to maintain the overall health of cells.Our results indicated that autophagy and apoptosis of hemocytes were important for maintaining homeostasis of hard clams under air exposure.The autophagic process of hemocytes may play an important role in the tolerance of hard clams to short-stages air exposure(low stress intensity),while apoptotic process plays an important role in the tolerance of hard clams to long-stage air exposure(high stress intensity).The significant increase in the rate of apoptotic hemocytes may be an important reason for the sudden decrease of ROS level.In addition,the results of this study suggested that granulocytes are the main site of ROS accumulation,and the main performers of autophagy and apoptosis.3.Transcriptional response of autophagy and apoptosis pathways in hemocytes of hard clams under air exposureAt the molecular level,our results suggested that autophagic and apoptotic process consist the important components of the tolerance mechanism to air exposure in hard clams.WGCNA results exhibited that the main biological processes occurred in the hemocytes during the first 20 under air exposure were the refolding of misfolded proteins in endoplasmic reticulum,lysosomal-mediated phospholipid degradation and protein synthesis and degradation.Notably,These biological processes were all closely related to autophagy.On the 20 th day of air exposure,the expression levels of a large number of genes encoding IAPs were significantly up-regulated,indicating that the anti-apoptotic system was activated,with the aim to delay the mass cell death.Additionally,our results showed that the expression levels of many important genes enriched in the autophagy and apoptosis pathways were significantly changed under air exposure.This study provides the valuable transcriptome resource for researches on hard clams,and adds a new understanding on the tolerance mechanism of intertidal shellfish from the perspective of autophagy and apoptosis of hemocytes.In terms of engineering practice,this study provided the survival curves of hard clam under air exposure at different temperatures(15℃,20℃ and 25℃),and determined the first death time and the lethal exposure time 50%.In addition,this study combined with the changes of autophagy and apoptosis related index of hemocytes and individual survival rate of hard clams,it was speculated that the transportation and preservation time of clam should be controlled within 5 days,and the long-distance transportation and preservation should not exceed 10 days.This study provides a scientific basis for the determination of transportation and preservation conditions of hard clams.