The Effect Of Nitric Oxide On The Metamorphosis And Immunity Of Mytilus Coruscus

Nitric Oxide(NO)is produced by the decomposition of L-arginine by Nitric Oxide Synthase(NOS),and it is a second messenger.In previous studies,NO is a negative regulator of marine invertebrate larvae,and it is also an important regulatory factor in the immune system.But it's effect on the metamorphosis and immunity of mussels has not been reported.Three methods of pharmacology,molecular biology and immune stimulation were used to explore the role of NO in metamorphosis and adult immunity of Mytilus coruscus.1.Effects of NO donors and NOS inhibitors on metamorphosis of M.coruscus.The results showed that the NOS inhibitors:aminoguanidine hemisulfate,S-methylisothiourea sulfate,L-NG-nitro arginine,7-nitroindazole all induced the metamorphosis of the larvae of mussel in the 24 h exposure and continuous exposure experiments,and the aminoguanidine hemisulfate had the highest inducing activity,and the post larvae rate reached 49%in the continuous exposure experiment.NO donors had different degrees of inhibition on the metamorphosis of larvae of M.coruscus.In conclusion,NO can inhibit the metamorphosis larvae of M.coruscus.This study provides an effective theoretical basis for the study of the regulation mechanism of the metamorphosis of M.coruscus.2.Temporal and spatial expression of nitric oxide synthase gene in M.coruscus.In order to further explore the role of nitric oxide in the metamorphosis of larvae of M.coruscus,we studied the temporal and spatial expression of nitric oxide synthase gene in M.coruscus.The NOS gene was cloned by RACE technique.The full length of NOS gene was 5091 bp and 4497 bp of open reading frame(ORF)encoding 357 amino acids.The qRT-PCR primers were designed based on the cDNA sequence of the NOS gene of M.coruscus in the transcriptional group.qRT-PCR technique was used to test the expression of the NOS gene in different developmental stages of the larvae(trochophore,D-shaped,umbo,pediveliger and juvenile),and tissue of adults(digestive gland,gonad,adductor muscle,foot,mantle,gill).NOS enzyme activity assay was used to verify the expression of NOS gene at the protein level.In the developmental stage,the NOS mRNA was highest in the stage of umbo stage larvae,and the expression of the pediveliger and the juvenile periods decreased in turn;the activity of NOS was the lowest in the trochophore,while the highest between D-shaped and umbo,then declined in the pediveliger and the juvenile,which was consistent with the NOS gene expression at the developmental stage.Thus,NOS may be involved in the transformation process of shell morphology and the formation and development of some organs in the larval stage.NOS gene may participate in the process of transformation of shell and the formation and development of some organs in the larvae,and the expression of NOS gene and the activity of NOS enzyme in the two critical stages(pediveliger and the juvenile)shells are lower than before,indicating that NO inhibits the metamorphosis of the larvae of M.coruscus,which was consistent with the pharmacological experimental results.In all tissues,the highest expression of NOS gene was found in gill,then were in the foot,digestive gland and the male gland.It indicates that the NOS gene is involved in the activities of respiration,filtration and digestion in the adult.In conclusion,NO inhibited the metamorphosis of larvae of M.coruscus.Taken together,the finding provides new insights on investigating the function of NO passway in shellfish.3.The influence of Vibrio splendidus on immune index and digestive enzyme activity of M.coruscusM.coruscus adults were challenged by V.splendidus in three concentrations(1×10~6cell/ml,1×10~7 cell/ml,1×10~8 cell/ml).The changes of immune index including nitric oxide synthase(NOS),nitric oxide(NO),superoxide dismutase(SOD)and malondialdehyde(MAD),and digestive enzyme activity including amylase and protease in digestive gland were determined during 72 h experiment period.The activities of NOS in foot,gill and digestive gland were measured after 48 h.Only the activity of NOS in the digestive gland was significantly different from the control group,and the activity of NOS increased with the increase of initial concentration of V.splebdidus.The activity of NOS of digestive in the experimental group was significantly higher than that of the control group after the challenge of V.splebdidu,and the level of NOS significant increased after 48 h.The content of NO was significantly higher than that in the control group.The content of NO increased first and then decreased,which had a similar trend with the activity of NOS.The activity of SOD was significantly higher than the control group after the challenge of V.splebdidu.The content of MAD in the experimental group was significantly lower than that in the control group after the challenge of bacteria.The activity of amylase in the experimental group was significantly lower than that in the control group.The activity of amylasethe descended first and then increased.The change of protease activity in the experimental group increased first and then decreased.This study indicates that V.splebdidu can promote the immune index and protease and inhibit amylasethe.The present study provides useful information for clarifying the immune mechanism of M.coruscus and vibrio-mussel interactions.