Effect Of Deep-sea Bacterial Biofilm On Larval Settlement And Metamorphosis Of The Mussel Mytilus Coruscus

Mytilus coruscus is an important economic shellfish in aquaculture in China.From the planktonic stage to the settlement stage,the larvae need to undergo settlement and metamorphosis before they can turn into plantigrades and then grow into adult mussels.Settlement and metamorphosis is an important stage in the growth and development of M.coruscus.In recent years,due to the artificial harvest of a large number of wild resources and the low rate of larval metamorphosis,the resources of M.coruscus have been greatly reduced.However,the market demand continues to increase,and there is a shortage of supply,so it is urgent to find the highly inducing active strain to solve the problem of aquaculture.This laboratory has proved that the biofilm(BF)of offshore marine bacteria can induce the settlement and metamorphosis of M.coruscus,so does the BF of deep-sea bacteria have the same effect? What are the factors that influence induction? This paper mainly studied the effect of deep-sea bacterial biofilm on the settlement and metamorphosis of M.coruscus larvae,determined the appropriate deep-sea bacterial biofilm culture temperature,and analyzed the mechanism of chemical signal regulating the settlement and metamorphosis of larvae.The main research contents are as follows:1.Effect of different deep-sea bacterial BFs on the settlement and metamorphosis of the mussel M.coruscus larvaeThe effects of deep-sea bacterial BFs on the larval settlement and metamorphosis of the mussel,M.coruscus,were investigated in the laboratory.Nine different deep-sea bacteria were selected to form BF at 18 °C,among which Virgibacillus sp.1 BF had the highest percentage of larval settlement and metamorphosis,and the induced activity of six deep-sea bacteria showed significant correlation with bacterial density.The Virgibacillus sp.1 BF cue that was responsible for inducing larval settlement and metamorphosis was further investigated.The BFs were treated differently(formalin,ultraviolet,organic solvents,antibiotics,temperature),and water-soluble metabolites were collected in the BF culture.Results found that the inducing activity and bacterial survival rate were significantly reduced in all treated-BFs.The water-soluble metabolites promoted(P<0.05)settlement and metamorphosis of larvae,but the synergistic effect with formalin-fixed BF significantly increased the inducting activity,indicating that two chemicals,surface substance,and water-soluble product,were needed to induce the settlement and metamorphosis of larvae.The biovolumes of extracellular products on the surface of BFs suggests that exopolysaccharides may play a role in the process of settlement and metamorphosis.2.Effects of biofilms of deep-sea bacteria under varying temperatures on larval metamorphosis of M.coruscusTemperature change is the primary problem for the adaptation of deep-sea bacteria.Effects of water temperature(4,18,25 and 37 °C)on deep-sea bacterial BFs and the subsequent effect on the metamorphosis of larvae of the mussel M.coruscus were studied.The confocal laser scanning microscopy technique was used to clarify the characteristics of BFs including BF thickness and the distribution of extracellular polymeric substances.The results showed that the BFs could promote the metamorphosis of larvae under four temperature conditions.Virgibacillus sp.1 showed the highest inducting activity(35%),with high α-extracellular polysaccharide secretion,the BF inducing activities on larval metamorphosis were correlated to the bacterial density(P<0.05).The temperature was significantly correlated with bacterial density,induction activity and BF thickness(P<0.05).The densities of bacteria in the BF of the two Pseudoalteromonas strains were significantly correlated with the induced activity(P<0.05).This study shows that temperature can affect the biological characteristics of BF of deep-sea bacteria,and then regulate larval metamorphosis.3.Regulation of alginate synthesis gene 07786 on biofilm and settlement and metamorphosis of the mussel M.coruscus larvaeBy constructing ?07786,the biofilm density,thickness,extracellular products were observed to investigate alginate synthesis gene 07786 effect on biofilm and settlement and metamorphosis of the mussel M.coruscus larvae.The results showed that Δ07786 biofilm decreased the settlement and metamorphosis of mussel larvae compared with wild-type biofilm.Analysis of the biological characteristics of Δ07786 biofilm revealed that the 07786 gene affected the biofilm formation by changing the biofilm bacteria density,biofilm thickness,and content of extracellular products,which in turn affected the settlement and metamorphosis of the mussel larvae.