Molecular Mechanism Of Brown Tide Mitigation Using Modified Clay

In recent years,harmful algal blooms(HABs)have become a global ecological disaster.How to mitigate HABs has become a critical research topic.With the advantage of low-cost,quick-effect,and non-toxicity,modified clay(MC)technology has become the most common emergency disposal method for HABs.Field studies on mitigating HABs with MC revealed that about 80% bloom organisms could be removed from water directly by flocculation,however,a bloom does not continue,even though the density of the residual cells in the water remains as high as 20% of the initial cell density approximately.In order to explore the effects of MC on residual cells and optimize the mechanism of MC mitigating HABs,the typical HAB(brown tide)organism Aureococcus anophagefferens was used as the model organism in this dissertation.Combined with scanning electron microscope(SEM)and atomic force microscope(AFM),molecular biology technique transcriptome sequencing(RNA-seq)and reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR)were used to study the molecular responses of residual cells to MC treatment,aiming to elucidate the indirect mechanism of MC controlling brown tide at the molecular level.The main results were as follows:1.The effects of MC on cell surface morphology of residual A.anophagefferens SEM technique was used to study the cell surface morphological characteristics of residual A.anophagefferens after treatment with MC.Besides,AFM technique was used to determine the clay surface properties before and after modification,such as,absorption site and adhesion,to further illustrate the reason that MC can inhibit residual cell growth effectively.The results showed that MC could break up some residual cells directly in a short period time,decrease the quantity of polysaccharide coated the cell membrane,and deform part of residual cells,resulting in cell “naked” and more vulnerable.And,the irreversible damage could inhibit proliferation.After modification by polyaluminium chloride(PAC),the number of absorption site and the value of adhesion force were increased on the clay particle surface,which intensified collisions and electrostatic effects between clay particles and algal cells followed by physical and chemical interaction enhancing,leading to stronger destruction on residual cells and inhibiting growth and proliferation effectively.2.The effects of MC on the transcription of residual A.anophagefferens RNAseq was used to study the transcriptional changes of various genes in residual A.anophagefferens cells treated with MC over time,and analyzed the response characteristics of various corresponding physiological processes comprehensively.The results showed that MC treatment caused oxidative stress to residual cells resulting in gene differential expression in them,among which,genes involved in the light reaction process were the most affected.In addition,numerous differentially expressed genes(DEGs)(p< 0.05)were enriched in the physiological processes and cell structures related to stress adaptation and detoxification,signal transduction and communication,membrane and cytoskeleton,genetic information processing,and the cell cycle.These results illustrated that after treatment with MC,physiological processes were affected in residual A.anophagefferens,and growth hindered.It was also found that the differential expression of these genes occurred in different time point: genes encoding membrane transporters and wound healing response proteins were up-regulated at 3 h after MC addition;however,until 24 h,basic metabolism genes responded to MC treatment,with the exception of photosyndissertation genes.These data suggested that plasma membrane and cytoskeleton were the first sites of damage.Collisions and charge neutralization between MC particles and residual cells may be the main cause accounting for the physiological response.3.The transcription dynamics of typical functional genes under the effects of MC In order to study the effects of MC on the dynamics of transcriptional expression in residual cells,40 typical functional genes were selected and RT-qPCR technology was used to measure the differential expression of these genes within 6 d after MC addition.These functional genes are mainly involved in five physiological processes,including anti-oxidation,photosyndissertation,phospholipid syndissertation,programmed cell death,and the cell cycle.It was found that the differential expression of every functional gene category exhibited a "V" shaped pattern in dynamics(the upregulated expressions of every functional gene category occurred twice in the time series with a low expressed turning point)reflecting that there were two main phases for MC inhibiting the growth of residual cells: one is the oxidative stress process which disturbed the normal physiological activities,resulting in cell lysis for damaged residual cells;the other is the programmed cell death(PCD)process induced by infochemicals released by lysed cells,which accelerated the death of residual cells and further controlled brown tide.The main innovation of this dissertation was: using molecular biology technique RNA-seq and RT-qPCR to explore the underlying mechanism of MC inhibiting residual cell growth from the aspect of transcription for the first time,found that MC could induce PCD characters of residual A.anophagefferens in molecular biology,revealed the indirect effect of MC controlling brown tide,optimized the mechanism of MC mitigating HABs.Above all,start with the cell surface morphological changes,this dissertation carried out the study of the effects of MC on residual A.anophagefferens from the level of molecular biology,found that collisions and charge neutralization between MC particles and residual cells are the main external factors inhibiting the growth of residual cells,whereas the decreased polysaccharide coated the cell membrane as well as oxidative stress and even PCD occurred on the residual cells are main internal factors accounting for growing and proliferating slowly.This dissertation revealed the effects of MC on residual cells,optimized the mechanism of MC controlling HABs,provided vital evidence for MC mitigating HABs effectively,and had important scientific significance for further research and development efficient MC species.