The Modulation Role Of Serotonergic System In Pacific Oyster Crassostrea Gigas In Response To Air Exposure

As an important component of the neuroendocrine system,serotonin?5-HT?system plays a major role in the regulation of various biological processes,including immune responses,energy metabolism,emotional behaviours,growth,reproduction,and stress responses to the changing environment,in both vertebrates and invertebrates.The 5-HT neuroendocrine system could be activated right after receiving an environmental stress or a stimulation,resulting in the synthesis and release of 5-HT to modulate the subsequent physiological processes responsive to these threats.By far,few researches have been investigated upon the molecular components of 5-HT neuroendocrine system,and the roles of 5-HT neuroendocrine system under air exposure in molluscs.In the present study,the molecular components and expression features of 5-HT neuroendocrine system,and the response pattern of 5-HT neuroendocrine system under air exposure were explored in oysters for better understanding of the modulation of 5-HT system under air exposure.Four 5-HT receptors?Cg5-HTR?including Cg5-HTR1,Cg5-HTR2,Cg5-HTR3 and Cg5-HTR4 were cloned from Pacific oyster Crassostrea gigas,with seven transmembrane regions.The cDNA sequence of Cg5-HTR1 was 1443 bp,encoding a polypeptide of 480 amio acids.The cDNA sequence of Cg5-HTR2 was 1239 bp,encoding a polypeptide of 412 amio acids.The cDNA sequence of Cg5-HTR3 was 1254 bp,encoding a polypeptide of 417 amio acids.The cDNA sequence of Cg5-HTR4 was 1008 bp,encoding a polypeptide of 335 amio acids.The mRNA transcripts of four Cg5-HTR were expressed in hepatopancreas,haemocytes and gonad,with the highest expression level detected in hepatopancreas.The mRNA transcript of Cg5-HTR1 was also expressed in mantle,while Cg5-HTR2 was expressed in adductor muscle.And,the mRNA transcripts of Cg5-HTR3 and Cg5-HTR4 were widely expressed and also determined in gill,adductor muscle and mantle.In addition,the tissue distribution of 5-HT was further investigated with immunoflouroscence.5-HT was mainly distributed in visceral ganglion,the edge of mantle and the gaps among hepatic tubules of hepatopancreas,but randomly and weakly expressed in gill,the exterior structure of labial palps and gonad.No positive signal was detected in adductor muscle.Furthermore,after recombinant plasmid of Cg5-HTR3 was transfected into Hela cells,the level of intracellular Ca²⁺ was significantly increased after the incubation of 5-HT.However,the effect could be inhibited by two different type antagonists?Way-10063 and Metagoline?of 5-HTR.5-HT concentration increased significantly in haemolymph of the oysters after they were exposed to air,especially during the late stage of the exposure.Meanwhile,the change of 5-HT concentration in mantle was detected after air exposure.The positive signals of 5-HT were firstly appeared in the edge of mantle under normal condition,and the signals became stonger along with the duration of air exposure.Moreover,the mRNA expressions of the four Cg5-HTR were also increased significantly after air exposure.Specifically,the mRNA expressions of Cg5-HTR1 and Cg5-HTR3 in haemocytes were increased significantly at late stage,while Cg5-HTR2 mRNA was increased significantly at early stage.The mRNA expression of Cg5-HTR4 was increased during the early stage after treatment,then decreased and restored to the initial level during the late stage.The apoptosis rate of haemocytes was remarkably declined,while the activities of superoxide dismutase?SOD?and nitric oxide synthase?NOS?in haemolymph were significantly increased after an injection of 5-HT into oysters under air exposure.Continuous air exposure would increase the apoptosis rate of haemocytes,SOD activity and H₂O₂ level in haemolymph,but decrease the NOS activity and the mRNA expression of CgTNF of haemocytes in oysters.In addition,once the oysters received an extra injection of 5-HT during air exposure,the SOD activity in haemolymph was significantly elevated,and the H₂O₂ level in haemolymph was astonishingly reduced.Meanwhile,the NOS activity in haemolymph was significantly elevated during the early stage of air exposure,and the apoptosis rate of haemocytes was remarkably reduced,while the survival rate of oysters was significantly increased during the late stage.In summary,molluscs such as oysters C.gigas have evolved a relatively complete 5-HT neuroendocrine system,and its major molecular components including 5-HT and 5-HTR were widely distributed in all the important tissues.When the oysters were exposed to air,5-HT neuroendocrine system could be activated and the levels of 5-HT in mantle and haemolymph,the mRNA expressions of the four Cg5-HTR were changed significantly after air exposure.The activated 5-HT could relieve the apoptosis and increase anti-oxidation ability of oysters,which played a key role in regulation of air exposure stress in oysters,and reduced mortality of oysters under air exposure.