| Marine biological fouling is one kind of serious problem when developing and using marine resources people will face.Due to the vast economics caused by it,it has attracted great attention from society in recent years.Environmentally friendly methods for preventing and controlling marine biofouling are a global consensus solution for marine biological fouling.There are many types of marine fouling organisms,including animals,plant and microorganisms.Among them,fouling animals are mainly marine invertebrates,and barnacles,as the typical representative fouling organism among them,have attracted the great attention of many researchers.Besides,the biofilm formed by marine benthic diatoms is also a significant member of the marine fouling community and an important basis for selecting habitats for large marine fouling animals.Extracellular polymeric substance(EPS)secreted by diatoms is a crucial substance that affects the formation of diatom biofilms.EPS and small molecular compounds from diatom biofilms guide marine invertebrate larvae to choose to attach and live a critical inducing signal for the ground.This article mainly focuses on the settlement and metamorphosis of marine invertebrate Amphibalanus(=Balanus)amphitrite larvae from planktonic life to sessile life and studies the benthic diatom organisms such as Amphora sp.The effect of membrane on the settlement and metamorphosis of the cyprid larvae of Balanus reticulatus.The main findings of the thesis are as follows:1.Biofilm plays an essential role in the settlement and metamorphosis of barnacle larvae.The biofilm formed by different diatoms has distinctive induction effects on the settlement of barnacle cyprid larvae.In this paper,two methods of biofilm supernatant larval settlement experiment and Double Disk experiment were used to study the effect of five species of marine benthic diatoms,including Amphora sp.,Stauroneis acuta,Chaetoceros gracilis,Phaeodactylum tricornutum Bohlin,and Nitzschia closterium.The influence of the settlement metamorphosis of the cyprid larvae of barnaus.The results showed that among the five diatom biofilms,the benthic diatoms that can induce the settlement of cyprid larvae are Amphora sp.,Stauroneis acuta,and Phaeodactylum tricornutum.Chaetoceros gracilis and Nitzschia closterium have no noticeable induction effects on them.During the initial period of 24h-48h,the settlement rate of cyprid larvae induced by Amphora sp.and Stauroneis acuta was the highest;at 72h of the mid-set period,the settlement rate of cyprid larvae caused by Amphora sp.and Stauroneis acuta still maintained a high level;to at 96h at the end of settlement,the larval settlement rate induced by Amphora sp was the highest,reaching 88.06%,followed by the larval settlement rate induced by Stauroneis acuta,reaching 83.14%.Therefore,this study believes that the marine benthic diatom with the inducing activity and the best activity for the settlement of cyprid larvae is Amphora sp,followed by Stauroneis acuta2.EPS polysaccharide of diatom biofilm significantly affects the settlement and metamorphosis of barnacle larvae.Diatom biofilms secrete EPS,which is divided into Soluble EPS(SL-EPS)and Bound EPS(B-EPS).SL-EPS mainly exists in the supernatant of the benthic diatom culture system,and is the first signal that barnacle larvae come into contact with when they explore their settling sites.In this paper,the crude polysaccharide in the supernatant of benthic diatoms and the de-sugar supernatant was obtained by the alcohol precipitation method.The larval settlement test found that the larvae settlement rate in the treatment group containing crude polysaccharide was significantly higher than that in the de-sugar supernatant Group,indicating that the polysaccharide in SL-EPS has a substantial inducing effect on the settlement of barnacle larvae.By PMP derivatization and HPLC-DAD detector,10 kinds of monosaccharides are detected in Amphora sp.SL-EPS,in descending order of molar ratio:Glc-A,Gal,Glc,Fuc,Rha,Glc-N,Xyl,Glc-Nac,Man,Gal-A.There are 9 monosaccharides in Stauroneis acuta SL-EPS,in descending order of molar ratio:Fuc,Gal,Glc-A,Glc,Rha,Man,Glc-N,Glc-Nac,Xyl.By analyzing the effects of different monosaccharides on the settlement and metamorphosis of barnacle larvae,it is found that different monosaccharides have different effects on the settlement and induction of cyprid larvae.In this study,11 standard monosaccharides were selected,and four monosaccharides with the best-inducing activity were selected for subsequent orthogonal experiments.The four monosaccharides were Man,Xyl,Fuc and Glc.Through orthogonal experiments,the effect of the combination of Man,Xyl,Fuc and Glc monosaccharides on the settlement and metamorphosis of barnacle larvae was studied.It was found that the addition of monosaccharides had a significant effect on the settlement of barnacle cyprid larvae.The optimum concentrations of Man,Fuc and Glc are 5.00×10-8 mol/L,1.90×10-7 mol/L and 0.31×10-8 mol/L,respectively.Xyl has little effect on larval settlement.The addition of monosaccharides not only affects the settlement rate of cyprid larvae,but more importantly,it can shorten the time course of larval settlement.The above results indicate that the benthic diatom biofilm releases SL-EPS to the outside world.The sugar signal carried therein may be an essential basis for barnacle larvae to identify and select habitat.3.The small molecules such as nucleosides and histamine that the diatom biofilm may release have a significant inducing effect on the settlement and metamorphosis of barnacle larvae.This article analyzed the impact of nucleosides on the settlement and metamorphosis of barnacle larvae and found that adenosine,uridine and guanosine had an inducing effect on the settlement of cyprid larvae.Orthogonal experiments of these three nucleosides and histamine showed that the concentration was 5×10-8mol/L adenosine,5×10-9mol/L uridine and 5×10-11mol/L.Guanosine can significantly increase the settlement rate of cyprid larvae,and the inducing activity is in descending order:A>>G>U>H.In this paper,n-butanol and n-hexane were used to extract histamine in the supernatant of the algae liquid.The OPA derivatization method and the HPLC-fluorescence detection method were used to analyze the presence of histamine in five diatom biofilms.The results found that the tested biofilms of Amphora sp.,Stauroneis acuta,Chaetoceros gracilis,Phaeodactylum tricornutum Bohlin,and Nitzschia closterium contain histamine,and its content in descending order is Amphora sp.,Stauroneis acuta,Nitzschia closterium,Phaeodactylum tricornutum Bohlin,Chaetoceros gracilis.Moreover,this result is consistent with the order of the inducing effect of each biofilm on larvae settlement.4.Transcriptome analysis of the histamine-induced mechanism of settlement and metamorphosis in barnacle larvae.Transcriptomics sequencing methods were used to study the changes in gene transcription levels under histamine-induced and histamine-antagonism.And try to analyze its signal pathways.Use NCBI non-redundant(nr),NCBI nucleotide sequences[nt],Clusters of Orthologous Groups of proteins(COG),Protein family,Swiss-Prot,Kyoto Encyclopedia of Genes and Genomes(KEGG),Gene Ontology(GO)(Total 7 databases)to annotate these single genes.Use two methods to screen differentially expressed genes:(1)different expression of genes under histamine-induced and histamine-antagonism.(2)Due to histamine can induce Cyprid larva settlement and histamine antagonist can inhibit Cyprid larva settlement,four filter conditions are set up.As a result of the second screening method,19 genes were selected.Then 19 genes were divided into 6 categories:structural proteins,spider silk proteins,energy metabolism proteins,cement proteins,glycosyl proteins,and unannotated proteins.Among them,the chitin protein gene in the structural protein may be related to the settlement and metamorphosis of the Cyprid larvae to develop a chitin shell.The spider silk protein gene may be associated with the barnacle mucus secreted by the Cyprid larvae.The energy metabolism protein gene is fat in Catenin receptor protein gene ADIPOR1 is closely related to energy metabolism in signal pathways,including lipid metabolism and sugar metabolism.QPCR verification results showed that ADIPOR1 expression of the histamine-induced group was significantly higher than the control group.In contrast,in the histamine antagonist group,it was significantly lower than the control group.And the result corresponds to the filter conditions of gene selection.In addition,Cyprid larvae were treated with 5×10-8mol/L histamine solution and 5×10-7mol/L histamine antagonist solution.Then test the AMPK enzyme activity.As a result,the AMPK enzyme activity of Cyprid larvae was significantly higher than that of the control group,and the AMPK enzyme activity concentration of Cyprid larvae treated with histamine antagonist was significantly lower than that of the control group.This result is corresponding with the result of the ADIPOR1 expression.Combined with the regulation of the gene ADIPOR1 in the AMPK signaling pathway,this study believes that the regulation of the sugar metabolism of the gene ADIPOR1-AMPK pathway may play an essential role in the settlement and metamorphosis of Cyprid larvae.In summary,this article studied the SL-EPS of macromolecular polysaccharides,small molecule histamine,nucleoside and other substances released by benthic diatom biofilms such as Amphora sp.It analyzed their inducing effect on the settlement of barnacle cyprid larvae.Impact.The study results confirmed that the sugar signal,histamine,and nucleoside substances of SL-EPS polysaccharide have a significant inducing effect on the settlement and metamorphosis of barnacle larvae.Transcriptome sequencing showed that after treatment with histamine and antihistamine,gene expression in larvae was significantly different.The ECM receptor interaction pathway and focal adhesion pathway were closely related to the settlement metamorphosis of barnacle cyprid larvae.Chitin receptor protein The up-regulation of genes such as irritin receptor protein(RYR2)and the regulation of lipid metabolism by the gene ADIPOR1-AMPK pathway are essential signs of larval settlement metamorphosis.These results provide more insights for further research on the settlement and metamorphosis mechanism of barnacle larvae. |
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