The Regulation Of Lactate Mediated Hypoxia Inducible Factor(EsHIF-1α) On The Response Of Haemocytes To Hypoxia Stress In Eriocheir Sinensis

Chinese mitten crab(Eriocheir sinensis)has a delicious taste and rich nutritional value,which is also one of important crustacean aquaculture species.During the breeding and commercial transportation of Chinese mitten crab,the changes of water environment,high-density breeding and long-distance transportation often result in the hypoxic stress.In order to explore the mechanism adapting to hypoxia stress in E.sinensis,the hypoxia-inducible factor Es HIF-1α was identified,and its role in regulating the response of haemocytes of Chinese mitten crab under hypoxia stress was investigated,the functions of Es HIF-1α and lactate in the process of haemocytes adapting to hypoxic stress was analyzed,and the potential menchanism of the change of haemocyte ratio under hypoxic stress was revealed.Finally,the internal relationship between lactate stimulation and haemocyte function was also clarified.The regulatory network of Es HIF-1α and lactate regulation involved in the function of haemocytes of E.sinensis to cope with hypoxia stress was preliminarily outlined.The main results and conclusions of the present study are as follows:1.The open reading frame length of the hypoxia-inducible factor Es HIF-1α is3174 bp,and the encoded amino acid sequence is 1057 aa.Sequence alignment,domain prediction and phylogenetic tree analysis showed that HIF-1α is a highly conserved sequence,which contains HLH,PAS and PAC domains.Its position in the evolutionary tree is similar to that of Scylla paramamosain and Penaeus vannamei,belonging to Decapod crustaceans.The protein level of Es HIF-1α gene in haemocytes of Chinese mitten crab was significantly increased after hypoxia stress for 3 h compared with normoxia,and the expression level was the highest at 12 h,which was 9.5 folds of normoxia(p < 0.01).After 24 h,it gradually decreased,but it was still higher than normoxia.Flow cytometry showed that the classification of haemocytes also changed significantly under hypoxia stress.The proportion of haylinocytes increased significantly after hypoxia stress and reached the highest after 24h(6.0 folds of the proportion of haylinocytes in normoxia group,p < 0.01),while the proportion of granulocytes decreased significantly under hypoxia stress and reached the lowest after24h(0.2 folds of the proportion of granulocytes in normoxia group,p < 0.01).2.The gene expression of different types of haemocytes in Chinese mitten crab was different,among which the expression of HIF-1α in haylinocytes was the highest,while the cell cycle and energy metabolism genes such as GLUT and AMPK in semigranulocytes and haylinocytes were higher.It is speculated that granulocytes may be the most active cells in the hemocytes of Chinese mitten crab,while haylinocytes belong to immature and younger haemocytes.The IOX4(activator of Es HIF-1α)significantly increased the proportion of haylinocytes and decreased the proportion of granulocytes,while the injection of KC7F2(inhibitor of Es HIF-1α)inhibited the increase of the proportion of haylinocytes and the decrease of the proportion of granulocytes in haemocytes under hypoxia stress.Activation of Es HIF-1α also activated hemocyanin gene expression in haemocytes.It is suggested that Es HIF-1αcan participate in regulating the proportion of haemocytes and increasing the synthesis of hemocyanin in haemocytes to achieve the adaptation of Chinese mitten crab to hypoxic stress.3.The increase of lactate level in hemolymph can significantly affect the expression of related genes such as Es HIF-1α,Es GLUT,Es Caspase and other genes in hemolymph,and affect the typing of hemolymph,suggesting that the increase of lactate under hypoxic stress may play a huge role in the adaptation of Chinese mitten crab to hypoxic stress,but After injection of(R)-GNE-140(inhibitor of LDH),the genes expression of haemocytes showed the opposite trend.In addition,the results showed that the survival rate of Chinese mitten crab injected with lactate under hypoxic stress was higher in the early stage of acute hypoxic stress.In summary,our study indicates that Es HIF-1α and lactate can regulate the function of haemocytes by regulating the ratio of haemocytes in Chinese mitten crab under hypoxia stress,thereby improving the adaptability of Chinese mitten crab respond to acute hypoxia stress.