| Ocean acidification（OA）alters the chemical equilibrium of seawater and hence affects the ion homeostasis such as calcium homeostasis in marine organisms.OA has been demonstrated to disrupt the calcification process of bivalves by affecting the homeostasis of calcium levels.The oyster Crassostrea gigas is an important marine calcifying species living in the estuaries and intertidal zones,which is widely distributed worldwide with economic and ecological significance.In this study,C.gigas was taken as the research object to observe the effect of OA on its newborn calcified shells,investigate the change of calcium homeostasis in its mantle under OA,and analyze the molecular characteristics of a pivotal calcium regulatory gene CgCaM in C.gigas and its expression patterns in different mantle folds under OA.1.OA affects the ultrastructure and calcium content of the inner surface of newborn calcified shells in C.gigasThe ultrastructure of the inner surface of newborn calcified shells in C.gigas was observed by scanning electron microscope（SEM）.Compared with the control group（p H 8.1）,it was found that after acidification treatment（p H 7.8）for 3 days,the aragonite sheets were loosely arranged and holes appeared at the junction of its margin;after acidification treatment for 7 days,the margin of aragonite sheets became smooth;after acidification treatment for 14 days,the margin became more smooth and the cracks in the surface increased of the aragonite sheets;after acidification treatment for 28 days,the aragonite sheets became smaller and appeared dissolved,and the crystal form became irregular.The relative calcium content of the inner surface of newborn calcified shells in C.gigas was analyzed by energy dispersive spectrometer（EDS）.It was found that after acidification treatment for 3,7 and 14 days,no significant change of calcium content was observed;after acidification treatment for 28 days,the calcium content was significantly decreased.2.OA affects the calcified shells formation of C.gigas by affecting the calcium homeostasis in its mantleIn the whole mantle,the Ca2+concentration was significantly increased after acidification treatment for 7 days,significantly decreased after acidification treatment for 14 and 28 days,and had no significant changed after acidification treatment for 3 days.In the different fold of mantle,the highest Ca2+concentration was both found in outer fold（OF）.The Ca2+concentration was significantly increased in inner fold（IF）,middle fold（MF）and OF after acidification treatment for 3 days,significantly increased in IF and MF after acidification treatment for 7 days,significantly decreased in IF and increased in MF after acidification treatment for 14 days,significantly increased in IF and OF after acidification treatment for 28 days.In the whole mantle,the alkaline phosphatase（ALP）activity was significantly decreased after acidification treatment for 7 days,significantly increased after acidification treatment for 14 and 28 days,and had no significant changed after acidification treatment for 3 days.In the different fold of mantle,the ALP activity was significantly decreased in IF and MF after acidification treatment for 3 days,significantly decreased in IF after acidification treatment for 7 days,and had no significant changed in IF,MF and OF after acidification treatment for 14 and 28 days.3.CgCaM is involved in the regulation of calcium homeostasis in mantle under OAA calmodulin（Ca M）was identified from the genome of C.gigas,and designated as CgCaM.The complete ORF of CgCaM was of 474 bp,encoding a polypeptide of 158 amino acids.There were four continuous typical EF-hand domains in CgCaM.The deduced amino acid sequence of CgCaM shared high identity with those Ca Ms in other species.In the phylogenetic tree,CgCaM was firstly clustered with calmodulin-A-like protein of Crassostrea virginica and then joined into the invertebrate group.Ca2+dependent electrophoretic migration assay showed that r CgCaM protein had calcium-binding activity.The m RNA transcripts of CgCaM were detected in all the tested tissues with the highest expression level in mantle,followed by the gill and labial palp.In the whole mantle,the m RNA expression level of CgCaM was significantly decreased after acidification treatment for 3 days,significantly increased after acidification treatment for 7 days,and had no significant changed after acidification treatment for 14 and 28 days.In the different fold of mantle,the m RNA expression level of CgCaM was significantly decreased in OF after acidification treatment for 3 days,significantly increased in IF and OF and decreased in MF after acidification treatment for 7 days,significantly decreased in IF and MF after acidification treatment for 14 days,significantly increased in MF and decreased in OF after acidification treatment for 28 days.The result of in situ hybridization showed that the positive hybridization signals of CgCaM were observed in epithelium of mantle,after acidification treatment,the positive signals were weakened,no obvious positive signals were observed in the outer epithelium of MF.In conclusion,OA had two-fold effects on the calcified shells formation of C.gigas under OA:reduced calcification rate after the short-term acidification treatment;calcified structures dissolution after the long-term acidification treatment.OA may affect the calcified shells formation of C.gigas by altering the calcium homeostasis in its mantle,among which CgCaM was involved in the regulation of calcium homeostasis in the mantle under acidification treatment.Furthermore,we inferred that distinct mantle folds were responsible for different function in the regulation of calcified shells formation under OA.Relevant studies may deepen our understanding of the mechanism of calcium homeostasis regulation in C.gigas under OA,further analyze the adaptation mechanism of bivalves to OA,and provide theoretical support for healthy aquaculture of C.gigas under OA. |
