| In recent years,the wide application of rare earth elements(REEs)has led to its increasing in the environment,resulting in increasingly hazards.In the previous studies,the effects of REEs was focused on terrestrial plants and its environment.Nevertheless,there are very few reports on the effect of the REEs on the safety of aquatic organisms and the environment.The outbreak of cyanobacterial blooms is not only related to their strong adaptability and reproduction,but also to the nutritional status.Therefore,Microcystis aeruginosa,the dominant species of cyanobacteria blooms in Taihu Lake,was selected as the object of this study.Main goal of the study is to explore the effect of exogenous REEs on the safety of an aquatic environment by choosing the lanthanum [La(III)] as the representative of REEs,and to simulate the pollution of La(III)in water at different trophic levels.An optimized combination of the methods is consisted of liquid chromatography-mass spectrometry,real-time fluorescence quantification and confocal laser scanning microscope,etc..Such combination of the methods was used here for initial investigation of the effects of an exogenous Lanthanum on the growth of M.aeruginosa,the content and types of microcystins,the key enzymes of microcystin's synthesis(non-ribosomal peptide synthetases,?-isopropylmalate synthase,prephenate dehydrogenase,tryptophan synthetase and argininosuccinate synthetase)and their genes(leuA,tyrA,trpB,argG,mcyA,mcyB and mcyC),elements,and endocytosis.The purpose of this study was to investigate the impact of the growth and microcystins(MCs)of M.aeruginosa induced by REEs,and explored the internal cause of the changes.The obtained results were shown as follows:(1)The La(III)affected the population growth of M.aeruginosa.Under normal nutritional status,the population growth of M.aeruginosa is slow within 0-8 days(adaptation period).After the adoption,in 8-20 days,M.aeruginosa grew faster and entered the logarithmic growth phase.The population growth rate reached the maximum,while the number of cells increased exponentially.Within 20 to 28 days,the cell density reached its maximum,cell division and death reached a dynamic equilibrium state,and the growth cycle entered a stable stage.After 28 days,the cell density began to decrease,showing a negative growth trend,and the growth entered a decline period.The growth pattern of M.aeruginosa was in a consistence with the pattern at a normal nutritional level.The effect of the exogenous La(III)on the M.aeruginosa population growth was influenced by the doses of the La(III).The cell density,intrinsic growth rate,and environmental capacity of M.aeruginosa after exposure to 2.0 ?M La(III)increased by 4.8%,11.3%,and 16.5%,respectively,compared to the control.On the other hand,high-dose La(III)(40.0 ?M)inhibited the M.aeruginosa population growth.The cell density and the intrinsic growth rate of population decreased by 16.6% and 12.6%,respectively,compared to the control.Low-dose La(III)(2.0 ?M)increased the maximum biomass of M.aeruginosa at both low and high nutrient levels by 10.7% and 17.0%,respectively,compared to the control.The intrinsic growth rate at low and high nutrient levels increased by 0.463% and 0.346%,respectively,compared to the control.Higher dose of the La(III)(40.0 ?M)decreased the maximum biomass,as well as the intrinsic growth rate.Therefore,lower concentration of the La(III)(2.0 ?M)could promote the population growth at the different nutritional levels,while high-dose La(III)(40.0 ?M)would inhibit.(2)With the population growth,La(III)exposure affected the content and composition of cellular MCs.MC-LR,MC-YR and MC-LW in algae cells,which increased gradually during the growth at normal nutritional status.The contents of the MC-LR,MC-YR,and MC-LW were stable during the stable period.However,their contents decreased during the population growth in the decline period.Similar phenomena were also noticed at different nutritional levels.The population growth at investigated nutritional levels was the same as that at the normal nutritional level.In the same culture period,MC-LR content was higher at high nutritional level comparing to the content at low nutritional level,while MC-YR and MC-LW contents were lower at high than at low nutritional level.Low-dose La(III)(2.0 ?M)increased the contents of MC-LR,MC-YR,and MC-LW in M.aeruginosa at normal nutritional level,while high-dose La(III)(40.0 ?M)inhibited.When the growth entered a stable period,the concentrations of MC-LR,MC-YR,and MC-LW increased by 12.0%,32.1%,and 57.5%,respectively,compared to the control after the exposure to the 2.0 ?M of La(III).When the dose of La(III)increased to 40.0 ?M,the concentrations of MC-LR,MC-YR,and MC-LW decreased by 20.6%,41.7%,and 58.8%,respectively,compared to the control.Low-dose La(III)(2.0 ?M)also increased the contents of MC-LR,MC-YR,and MC-LW at low and high nutrient levels,while high-dose La(III)(40.0 ?M)decreased their concentration.(3)The intrinsic mechanism of La(III)affecting MC-LR,MC-YR and MC-LW synthesis was revealed from the key enzyme activities and gene expression of MCs synthesis.There was no significant difference in the key enzyme activities and gene expression of MCs synthesis between low and high nutrient levels,but it showed significant differences under different concentrations of La(III).Low-dose La(III)(0.2 and 2.0 ?M)increased the activities of MCs synthases(non-ribosomal peptide synthetases,?-isopropylmalate synthase,prephenate dehydrogenase,tryptophan synthetase and argininosuccinate synthetase)and the transcriptional level of MCs synthase genes(leuA,tyrA,trpB,argG,mcyA,mcyB and mcyC)in M.aeruginosa.High-dose of La(III)(40.0 ?M)inhibited the activities and genes transcription of MCs synthase.The "low promotion,high inhibiting" effect induced by La(III)on the transcriptional levels of MCs synthase genes would affect the catalytic activities of MCs synthases in M.aeruginosa and change the production of MC-LR,MC-YR and MC-LW.(4)The mechanism of La(III)affecting the synthesis and composition of MCs was revealed from the perspective of the ecological stoichiometry and cytology.The concentration of MCs in cells was positively correlated with the molar ratios of C:P and N:P.The changes in the total MCs content were positively associated with the ratios of C:P and N:P in algal cells.In addition,the composition of MCs was dependent on the ratio of C:N in algal cells;for example,the percentage content of MC-LR decreased and the percentage content of MC-YR and MC-LW increased as the ratio of C:N in algal cells increased.After La(III)treatment,the fluorescent markers in M.aeruginosa fluoresced obviously in the algae cells,and activated the endocytosis of M.aeruginosa.The color of FM4-64 increased with the increase of La(III)dosage,and the activation of endocytosis was further enhanced.La(III)may affect the uptake of nutrient and mineral elements in algae cells through endocytosis,thereby affecting the growth of algae cells and the production of MCs.In addition,La(III)may activate the endocytosis of M.aeruginosa by clathrin-mediated endocytosis,affecting the growth of M.aeruginosa.The mechanism of the changes of population growth and MCs content of M.aeruginosa induced by exogenous La(III)was revealed in this study: La(III)entered algae cells through endocytosis and changed element uptake,resulting in the changes in gene transcription of MCs synthetases,affecting the catalytic activities of MCs synthetases,thereby affecting the population growth and the MCs contents of M.aeruginosa.The results provides a new insight into the mechanism of REEs effects on cyanobacteria and MCs,and further understanding the effect of REEs on cyanobacteria cells.It provides a reference for the objective assessment of environmental safety risk caused by REEs pollution in water body,and also provides datas support for the objective assessment of the stress response of harmful algae caused by REEs pollution in future environment. |
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