The Response And Adaptation Mechanism Of Pacific Oyster Crassostrea Gigas To Ocean Acidification

Ocean acidification?OA?has been demonstrated to have severe effects on marine organisms,especially marine calcifiers.Pacific oyster?Crassostrea gigas?is an important marine calcifier living in the estuaries and intertidal zones and also one of dominant aquaculture bivalves worldwide.In the present study,the molecular characteristics,expression profile and phylogeny relationship of three key enzymes,as well as their roles in regulation of physiological processes in response to OA were explored.The transcriptome libraries of haemocytes from the oyster C.gigas after short-time and long-time CO₂ exposure were sequenced in order to find new clues of the mechanism of mollusk response or adaptation to OA.CgCAII-1 possessed a conserved CA catalytic domain,with high similarity to invertebrate cytoplasmic or mitochondrial ?-CAs.Recombinant CgCAII-1 could convert CO₂ to HCO₃^-,which could be inhibited by AZ.The mRNA transcripts of CgCAII-1 in muscle,mantle,hepatopancreas,gill and haemocytes increased significantly after exposed to elevated CO₂.CgCAII-1 could interact with the haemocytes membrane proteins and the distribution of CgCAII-1 protein became more concentrated and dense in gill and mantle under CO₂ exposure.The intracellular pH?pHi?of haemocytes under CO₂ exposure increased significantly?p < 0.05?and CA inhibition reduced the pHi value.Besides,there was no increase in CA activity in gill and mantle after CO₂ exposure.The impact of CO₂ exposure on CA activity coupled with the mRNA expression level and protein translocation of CgCAII-1 provided evidences that CgCAII-1 could respond to ocean acidification and participate in acidbase regulation.The cDNA of CgCA was of 927 bp encoding a predicted polypeptide of 308 amino acids with a signal peptide and a CA catalytic function domain.The mRNA transcripts of CgCA were constitutively expressed in all tested tissues with the highest levels in mantle and hemocytes.During the early development period,the mRNA transcripts of CgCA could be detected in all the stages with the highest level in D-veliger larvae.Elevated CO₂ increased the mRNA transcripts of CgCA in muscle,mantle,hepatopancreas,gill and hemocytes significantly?p < 0.05?and induced the translocation of CgCA in hemocytes and mantle.Moreover,elevated CO₂ also caused the decrease of intracellular Ca²⁺ in hemocytes?p < 0.05?.The inhibition of CA by acetazolamide and suppression of CgCA gene via RNA interference could increase the intracellular Ca²⁺ in hemocytes?p < 0.05?.Besides,the decrease of intracellular Ca²⁺ content caused by Ca²⁺ reagent ionomycin could affect localization of CgCA in mantle tissue.The results indicated CgCA played essential roles in calcification and elevated CO₂ accelerated the mutual modulation between calcium and CgCA,implying reduced calcification rate and dissolved shells under OA.Soluble adenylyl cyclase?sAC?is an acid-base sensor in response to HCO₃^-and an intracellular source of cyclic AMP?cAMP?.In the present study,an ortholog of sAC was identified?designated as CgsAC?and the catalytic region of CgsAC was cloned and expressed.Similar to the native CgsAC from gill tissues,the recombinant CgsAC protein?rCgsAC?exhibited HCO₃^-mediated cAMP-forming activity,which could be inhibited by a small molecule KH7.After 16 days' CO₂ exposure treatment?pH=7.50?,the mRNA transcripts of CgsAC increased in muscle,mantle,hepatopancreas,gill,male gonad and haemocytes,and two truncated CgsAC forms of 45 kD and 20 kD were produced.Cytosolic CgsAC could be translocated from the cytoplasm and nuclei to the membrane in response to CO₂ exposure.Besides,CO₂ exposure could increase the production of cAMP and intracellular p H of haemocytes,which was regulated by CgsAC?p < 0.05?,suggesting the existence of a HCO₃^-/CgsAC/cAMP signal pathway in oyster.The elevated CO₂ could induce the increase of ROS level?p < 0.05?and decrease of haemocytes phagocytic rate?p < 0.05?,which could be inhibited by KH7.The apoptosis ratio in CO₂ exposure group?35.2 %?was significantly higher?p < 0.05?than that in the control group,and the increased apoptosis ratio induced by elevated CO₂ could be significantly inhibited?p < 0.05?by KH7.After CO₂ exposure,CgsAC was found to be co-localized with mitochondria in the cytoplasm,and the pro-caspase-3 was cleaved into two small fragments.Moreover,the activities of caspase-3 and caspase-9 also increased post CO₂ exposure and these increases could be inhibited by KH7.However,the activities of caspase-8 did not change significantly compared with that in the control group.After CO₂ exposure,the ATP content in the gill increased significantly?p < 0.05?and such increase could be also be inhibited by KH7.While the ATP content in purified gill mitochondria decreased significantly?p < 0.05?after CO₂ exposure,which was also inhibited by KH7.The results collectively suggested that CgsAC was an important acid-base sensor in oyster and the HCO₃^-/CgsAC/cAMP signal pathway might be responsible for intracellular alkalization effects on oxidative phosphorylation and innate immunity under CO₂ exposure.The transcriptome libraries of haemocytes from the oyster C.gigas after 7-day and 60-day CO₂ exposure were sequenced.Nine double-end fragment libraries were constructed and sequenced in the three groups,including blank,short and long group,and there were 72,504,095 single-end reads totally obtained with 28,027 genes identified.There were 501,and 2728 differentially expressed genes identified in the comparison of short versus blank,and long versus blank,respectively.Genes in the ‘membrane' and ‘ion binding' were enriched in the ontology of ‘cellular components' in both the short versus blank and long versus group and more genes related to transport were up-regulated in the long group than the short group.A set of significantly differently expressed genes belonging to the category ‘molecular function' were enriched in ‘heterocyclic compound binding'.In the short versus blank genes list,these genes were mainly ATP or GTP binding,such as cytoskeleton genes including actin,tubulin and dynein.In the long versus blank genes list,there were more genes involved in ATP or GTP binding and DNA or RNA binding including cytoskeleton genes and replication,transcription and translation related genes.‘Calcium ion binding' is another category highlighted by the GO enrichment analysis and varieties of calcium binding proteins?CBP?were up-regulated in the long group.Intracellular calcium concentration decreased significantly after short time CO₂ exposure and then increased to the level of the control group after long time exposure,while calcium content in the serum increased in both short and long group.Besides,the energy metabolism of the short and long groups were different.In the short group,both the glucose in serum and liver glycogen content decreased significantly,while in the long group the glucose in serum and liver glycogen content,were much higher than that of the blank group.The present results indicated that oyster displayed different mechanism in short-time CO₂ stress response and long-time CO₂ adaptation.In conclusion,key acid-base sensors including CA and sAC mediated acid-base regulation and downstream immune response and energy metabolism in oyster under CO₂ exposure.As important marine calcifier,oyster displayed different mechanisms in response and adaptation to elevated CO₂.