Effect Of L-arginine On Biofilm Formation And Larvae Metamorphosis Of Mytilus Coruscus

Mytilus coruscus is a large scale marine invertebrate,which mainly exists in southeastern coastal areas of China.At this stage,the scientific research on it mainly focuses on the following two aspects: first,the pollution prevention and treatment as marine fouling organisms,and second,the production benefits as economic species.Arginine is an important amino acid that can be metabolized by bacteria and used as an energy source and building block for protein synthesis.Research has found that arginine may inhibit bacterial biofilm formation by inhibiting cell c-di-GMP synthesis.At the same time,L-arginine(L-Arg)serves as the substrate for the synthesis of nitric oxide(NO),which has the effects of relaxing and dilating blood vessels.Previous studies in our laboratory have shown that the single biofilm formed by Pseudoalteromonas marina and its extracellular products such as polysaccharides,proteins,and lipids can induce settlement and metamorphosis of mussel larvae.However,as of now,it is unknown whether L-arginine affects the biofilm formation of P.marina and its regulatory effect on the metamorphosis of mussel larvae.This paper mainly discusses the effect of L-arginine on P.marina biofilm and larval metamorphosis by directly stimulating P.marina and mussel larvae,and draws the following conclusions:1.L-arginine inhibits biofilm formation of P.marina and its induction ability for larvae metamorphosis of Mytilus coruscusUsing P.marina biofilm as the control group,L-arginine solutions with final concentrations of 0,0.01,0.05,0.1,1,2.5,5,and 10 m M were co-cultured with P.marina to form biofilm,and its effect on larvae metamorphosis of mussel was studied.The results showed that when the concentration of L-arginine was more than 1m M,the bacterial density,film-forming ability,total sugar content,c-di-GMP level,and c-di-GMP synthesis related genes of biofilm bacteria were significantly reduced,leading to a significant decrease in the ability to induce metamorphosis in mussel larvae.This indicates that Larginine has an inhibitory effect on the biofilm formation ability,intracellular c-di-GMP level,and extracellular total sugar content of P.marina,and further inhibited the induction ability of biofilm on the larvae metamorphosis of mussel.2.L-arginine inhibits larval metamorphosis by promoting nitric oxide synthesis in Mytilus coruscusIn order to explore the molecular mechanism of L-arginine regulating larval settlement and metamorphosis,the eyespot larvae of mussel were exposed to L-arginine and nitric oxide synthase(NOS)inhibitor aminoguanidine hemisulfate(AGH)。The analysis of NO content,NOS gene expression and larval metamorphosis rate after joint exposure showed that L-arginine can increase NO content and upregulate NOS expression in the larvae,but significantly inhibit larval metamorphosis.The addition of AGH significantly inhibited the induction of NOS gene expression and NO production by Larginine,and led to a significant increase in larval metamorphosis rate.The results of knocking down the NOS gene expression of larvae by electrotransfection with si RNA showed there was no significant difference in the larvae metamorphosis rate.The above results indicate that exogenous L-arginine can synthesize NO through larval NOS,thus regulating its settlement metamorphosis.Mussel larvae can synthesize NO using exogenous L-arginine as a substrate and inhibit their own metamorphosis.In conclusion,L-arginine can inhibit the formation of P.marina biofilm by reducing the c-di-GMP content of bacteria and the total extracellular sugar content of biofilm,thereby inhibiting the regulatory effect of biofilm on larval settlement and metamorphosis.On the other hand,it can also serve as a substrate for NOS synthesis of mussel larvae to synthesize NO,thereby inhibiting larval settlement and metamorphosis.L-arginine inhibits the process of larval metamorphosis by regulating the formation of biofilms and the synthesis of NO,providing some basis for exploring the relationship between small molecule amino acids in the ocean,biofilm formation and the larvae settlement and metamorphosis.It will also provide new ideas and technical routes for the prevention and control of marine biological pollution in the future.