Mechanisms Of Physiological Responses And Transcriptional Regulation To Hypoxia Stress In Sinonovacula Constricta

Oxygen is necessary for the life activities of all aerobic organisms.The razor clam(Sinonovacula constricta)is a mudflat bivalve that is susceptible to aqueous humoral oxygen and dry dew stress.The razor clam has a strong hypoxia tolerance,and the investigation of the molecular mechanism of hypoxia tolerance and adaptation of razor clams is of great value for razor clam breeding and breeding.In this study,we investigated mechanisms of physiological responses and transcriptional regulation to hypoxia stress in S.constricta.The results of the study are as follows:1.Histological observations of razor clam after hypoxia stress showed that the overall gill tissue of razor clam contracted after hypoxia stress and the gill filament gap increased;gill filament width(GFW)and gill thickness(GT)became significantly smaller after hypoxia stress.After hypoxia,the basement membrane of the hepatopancreatic duct of razor clam appeared dissolved and irregularly cavitated,and the outline of the duct wall began to be blurred.The reduced glutathione(GSH)enzyme,alkaline phosphatase(AKP)and acid phosphatase(ACP)activity of the hepatopancreatic tissue of razor clam decreased significantly after hypoxia stress.2,Transcriptome sequencing analysis of gill and hepatopancreas tissues from six time points(0 h,6 h,12 h,24 h,36 h,48 h)under hypoxia(0.5 mg/L)stress in razor clam.GO enrichment of gill and hepatopancreas differential expressed genes(DEGs)showed that GO entries were mainly associated with organic matter metabolism,protein synthesis and transport,and protein degradation.KEGG enrichment analysis showed that several metabolic pathways such as tryptophan metabolism,fatty acid metabolism,and oxidative phosphorylation were significantly down-regulated,and autophagy-animal pathway was significantly up-regulated in hepatopancreas.Glycolytic pathway genes as well as PEPCK were significantly up-regulated after hypoxic stress.electron transport chainrelated genes(V-ATPase subunit H,CO1,Ndufs8)were significantly down-regulated in the TCA cycle.In the hypoxia environment,the genes of the razor clam part of the innate immune response were down-regulated(C1q-l4 and MRC2).However,TIR4 and TIR8 expression was up-regulated.Genes that promote cell proliferation,differentiation and apoptosis were up-regulated in gill tissue after hypoxia stress(Caspase2,Bcl2-L-7,PD-6,etc.).Significant module KEGG enrichment in WGCNA analysis revealed significant up-regulated of protein processes in ribosomes,endoplasmic reticulum,and spliceosome pathways.3,In this study,four key genes of HIF signaling pathway,HIF-1α,HIF-1β,PHD2 A and PHD2 B,were cloned from S.constricta,Sequence analysis showed that ScHIF-1 and ScPHD2 contains typical domains.Unlike other invertebrates,shellfish including razor clam,have two copies of PHD2.Expression level analysis showed that razor clam HIF-1 and PHD2 were expressed during the egg stage,and ScHIF-1α was expressed at higher levels than other genes in the six tissues examined,with the highest expression in the gills,while ScPHD2 was expressed at low levels in all six tissues.Under hypoxia stress,ScHIF-1α and ScPHD2 expressions were significantly up-regulated,but the 0.5 mg/L group was more up-regulated than the 2 mg/L group,while ScHIF-1β expressions were not significantly changed.Under air exposion stress,the expression of ScHIF-1β did not change significantly,while the expression of ScHIF-1α and ScPHD2 were significantly up-regulated,and the up-regulation was greater in the 4 ℃ air exposion group than in the 21 ℃ air exposion group.In this study,we initially analyzed the changes in tissue structure and enzyme activity of gill and hepatopancreas of razor clams after hypoxia stress and the changes in gene expression under hypoxia stress by fluorescence quantitative PCR,tissue sectioning and transcriptome sequencing,and conducted a preliminary investigation on the molecular mechanism of hypoxia adaptation of razor clam.This study provides a theoretical basis for the adaptation of razor clam to hypoxia and also provides theoretical support for healthy razor clam breeding.