| Mytilus coruscus is one of the most important shellfish economically in seawater.As a kind of Marine active peptide,Mytilus galloprovincialis peptide has the functions of providing nutrition,antibacterial,promoting biological metabolism and immune ability.As biofilm is one of the inducible factors for M.coruscus settlement,it is still unknown whether the effect of M.galloprovincialis peptide on biofilm will change its ability to induce settlement,and through which way M.galloprovincialis peptide will affect settlement.Two strains of Marine bacteria with different induced activities were selected: Pseudoalteromonas marina isolated from natural biofilm and Leisingera aquaemixtae isolated from the intestinal tract of M.coruscus.The formation of biofilm was directly stimulated by M.galloprovincialis peptide,so as to explore the influence of biofilm on the settlement of M.coruscus juvenile,and further explore how M.galloprovincialis peptide affects the settlement of biofilm and juveniles.The main results are as follow:1.Effects of M.galloprovincialis peptide on settlement of mussel juveniles induced by bacterial biofilmIn order to study the effects of marine bioactive peptide on the formation of marine bacterial biofilm and settlement of marine mussel juveniles,this study stimulated M.coruscus juveniles by agarogase encapsulation with different concentration of M.galloprovincialis peptide and observed its induction to M.coruscus juveniles’ settlement.Later on,two strains of marine bacteria with different settlement induction abilities were selected: P.marina and L.aquaemixtae.Different concentration of M.galloprovincialis peptide was added during biofilms forming process.The effects of M.galloprovincialis peptide on biofilm formation and biological characteristics were analyzed.At the same time,the effect of biofilm changes on M.coruscus juveniles settlement was studied.The M.coruscus juveniles settlement experiment results showed that M.galloprovincialis peptide could significantly induce M.coruscus juveniles settlement(p< 0.05).Besides,the effect of 1 g/L M.galloprovincialis peptide which was added during the formation of biofilm on settlement induction of M.coruscus juveniles was the most significant(p< 0.05)and the effect of 10 g/L M.galloprovincialis peptide on settlement of M.coruscus juveniles was lowest(p< 0.05).In the further experiment on biofilms,such as biofilm bacterial density counting experiment,biofilm thickness measurement experiment and laser confocal detection of membrane protein content experiment,it was found that 1 g/L M.galloprovincialis peptide induced the most significant effect(p< 0.05): 1 g/L M.galloprovincialis peptide was significantly increased the bacterial density and protein content of biofilms(p<0.05).;Although,10 g/L M.galloprovincialis peptide was significantly decreased the bacterial density and protein content of biofilms(p< 0.05).The results showed that M.galloprovincialis peptide could directly affect the settlement of M.coruscus juveniles,and indirectly affect the settlement of M.coruscus juveniles by affecting the biofilm formation level and protein content of marine bacterial biofilms.These results provided a new view for exploring the role of marine bioactive peptides in the settlement of M.coruscus juveniles and provided a new idea for studying the physiological function of bioactive peptides from marine shellfish.2.Molecular mechanism of M.galloprovincialis peptide on biofilm formationIn this study,three metabolites were found to be strongly related to metabolic pathways through metabolomics: purine metabolism and the anabolism of amino acids such as glycine,serine,threonine and arginine.In addition,arginine can affect the level of intracellular second messenger c-di-GMP,thus affecting biofilm formation and possibly affecting adhesion through further effects of c-di-GMP.In the intracellular metabolome,amino acid metabolism and carbon metabolism pathways of biofilm were significantly up-regulated after the addition of M.galloprovincialis peptide.It is speculated that the addition of M.galloprovincialis peptide can promote the metabolism of amino acid and carbohydrate in biofilm.Promote the accumulation of extracellular products of biofilm,promote the formation of biofilm and the settlement of M.coruscus juveniles.The expression of D-Ala-D-Ala was upregulated.Therefore,it is speculated that the addition of M.galloprovincialis peptide can up-regulate the synthesis of peptidoglycan,promote the formation of biofilm,and thus promote mussel settlement.In the extracellular metabolome,tryptophan pathway serotonin in extracellular metabolites was found to promote larval settlement and metamorphosis in M.coruscus,so it was speculated that it could affect settlement.However,the up-regulation of tryptophan indicated that the up-regulated tryptophan was being consumed and metabolized into serotonin,and thus did not have an inhibitory effect on biofilm formation.The down-regulation of alpha-linolenic acid did not inhibit biofilm formation.3.Molecular mechanism Study on settlement of M.coruscus juveniles directly induced by M.galloprovincialis peptide.This study was conducted to investigate the direct effect of M.galloprovincialis peptide on the settlement of M.coruscus.The results showed that the coating environment of M.galloprovincialis peptide had a significant ability to induce the settlement of M.coruscus juvenile.After injecting M.galloprovincialis peptide into the closed shell muscle of M.coruscus,it was found that M.galloprovincialis peptide promoted the expression of settlement related gene: mussel foot proteins and collagen gene,and its metabolism and immunoenzyme activity were significantly enhanced,indicating that M.galloprovincialis peptide could enhance the metabolism level and resist ability of M.coruscus to cope with adverse environment,and thus promote settlement.In conclusion,M.galloprovincialis peptide can affect settlement by influencing biofilm formation and extracellular protein secretion.It can also directly affect the genes related to byssus formation and metabolism and immune-related enzyme activities of M.coruscus and settlement,thus promoting settlement.These results preliminarily explained the molecular mechanism of the effect of M.galloprovincialis peptide on biofilm formation and M.coruscus juvenile settlement.At the same time,it improves the mussel breeding industry and expands the application field of Marine active peptides. |
PDF Full Text Request