Immune Response To Hypoxia Stress And The Mechanism Of HIF-1α Regulation In Blood Clam Tegillarca Granosa

The blood clam,Tegillarca granosa,mainly inhabits intertidal mudflats and experiences severe hypoxia due to tides,temperature changes,and human activities.This habitat makes the blood clam adapt well to hypoxia conditions.Studying the effects of hypoxia on the survival and immunity of blood clam conducive to further understand the molecular mechanism of blood clam in respond to hypoxia stress.For this purpose,we carried out the following studies:First,we compared the blood clam with Ruditapes philippinarum,Scapharca broughtonii,Crassostrea gigas,Sinonovacula constricta and Mactra veneriformis at dissolved oxygen concentration of 0.5 mg/L in seawater after 14 days of hypoxia stress.After 14 days of hypoxia stress,the survival rate of blood clam,S.Broughtonii and R.Philippinarum were 85%,20% and 58.75%,respectively,while all C.gigas,S.constricta,and M.veneriformis had a 100% mortality rate.Meanwhile,the blood clam survival rates in hypoxia groups were greater than 85% after 14 days of hypoxia stress,suggesting clams displayed strong hypoxia tolerance.Secondly,we investigated the immune-related indexes of blood clam at dissolved oxygen concentration of 0.5 mg/L.The results showed that the total number of hemocyte decreased significantly after five days of hypoxia stress(p < 0.01),only 66.3% of the control group.Hemoglobin concentration also decreased from 28.4 g/L to 25.6 g/L.Although the phagocytosis of hemocyte was not significantly affected by hypoxia stress for 72 hours,it was significantly inhibited after 120 hours(p < 0.01),and cell activities of hemocyte decreased significantly(p < 0.01).After 72 h and 120 h hypoxia stress,intracellular ROS levels were significantly inhibited by hypoxia stress(p < 0.01),which was 0.76 and 0.57 times that of the control group,respectively.Furthermore,the four immune-related genes(MAPK,NFκB1,Ras,and IKKα)expression was also downregulated.In conclusion,hypoxia inhibited the immune response of blood clam.Meanwhile,in order to study the adaptation mechanism of blood clam to the hypoxia environment,we obtained the key gene of the hypoxia signaling pathway,hypoxia-inducible factor(HIF),by screening the genome of blood clam.The bioactivity of HIF is mainly determined by the transcriptional activity of HIF-1α.The transcriptional activity of HIF is mainly determined by HIF-1α.The structural domain prediction results showed that there are four relatively conserved domains at the N-terminal of the HLH,PAS-A,PAS-B and PAC domains,respectively.The phylogenetic tree showed that the HIF-1α of blood clam was closely related to other Marine bivalves,but far related to insect.We also selected another key gene of the hypoxia signaling pathway,prolyl 4-hydroxylases(PHD),as well as hemoglobin(Hb)and myoglobin(Mb),which are related to oxygen carrying and storage.The expression of these genes under hypoxia stress was analyzed by q PCR.The expression of HIF-1αwas significantly different after 8 hours of hypoxia stress(p < 0.01).In 0.5 mg/L hypoxia group,the expression of HIF-1α in hemocyte was 10.45 times higher than the control group and the expression of HIF-1α in gill was 6.72 times higher than the control group.While in the 2.0 mg/L hypoxia group,the expression of HIF-1α in hemocyte and gill was3.86 and 5.09 times higher than the control group,respectively.The PHD expression pattern was similar to HIF-1α under hypoxia stress,and showed a trend of increasing followed by decreasing expression in hemocyte and gill.The expression levels of Hb I,Hb IIA,Hb IIB and Hb III decreased significantly under hypoxia stress(p < 0.01),and the expression levels of Mb I and Mb II were 5.43 and 2.24 times higher than those of the control group after 120 hours of hypoxia stress,respectively(p < 0.01).The results showed that the m RNA levels of HIF-1α,PHD,Mb I and Mb II genes were differentially induced to be expressed under hypoxia,and they play a certain role in the adaptation of blood clam to hypoxia.Finally,in order to investigate the regulation mechanism of HIF-1α of blood clam,potential hypoxia response elements(HREs)were identified by predicting the promoters of Hb and PHD.The promoters were cloned and constructed into a dual luciferase reporter gene vector and cotransfected into cells with HIF-1α transcription factor.The results showed that the dual luciferase activity of reporter genes PGL3-basic-HBI,PGL3-Basic-HBIIA,PGL3-Basic-HBIIB and PGL3-Basic-HBIII was not significantly different from the control,while the dual luciferase activity of reporter gene PGL3-basic-PHD increased by 2.44 times(p < 0.05).This indicates that HIF-1α of blood clam has transcriptional regulation of PHD,but not Hb I,Hb IIA,Hb IIB and Hb III.This is consistent with the negative feedback regulation of PHD2 by HIF-α binding to the HRE site in the C.gigas.