Exploration Of The Effects Of Light On Toxic Microcystis Strains And Comparison Of The Effects Of Microcystin-rr On Water Metabolism Between Rats And Mice

Microcystis is one of the most common blue-green algae that often appear in eutrophic lakes and relatively stagnant waters during the warm seasons.This kind of algae has drawn attention from the researchers and the public because some species can produce microcystins(MC)which can result in acute toxicity in domestic and wild animals in high doses and are regarded as a tumor promoter in low doses via long-term exposure to them by animals.Wuhan is a city with many lakes and situated in the center of China.The eutrophication in these lakes has continuously worsened the water quality during the past 40 years.In recent years in this region,the researches have not been carried out enough about the process of blooms,ecological damage,toxin-producing algal strains,toxicity mechanism of the toxins and health hazards,especially the basic research of the related physiological characteristics on toxic strains being very weak.Light is an important driver of the biological metabolism for the Microcystis,and the property,intensity and the illumination time of light may have different effects on the growth speed,metabolites,biomass accumulation of the cyanobacteria.Although short-term high-light intensity exposure may have the same total photons as long-term low-light intensity exposure based on physical measurement,their physiological and toxicological effects on an organism may not be the same.The light length is obviously different during the different seasons,but whether or not the longer the illumination is,the better Microcystis grows,or the more secondary metabolites such as toxins? There have been no reports or data that can be referenced yet.According to the reports,279 congeners of MC have been documented,but only a few MC have been systematically studied.Some researchers reported that not only microcystin-LR(MC-LR)could be found in waters but also microcystin-RR(MC-RR),in which the levels of MC-RR were frequently much higher,so research on MC-RR for its toxicity is of great importance.At present,most researchers focus on the histological,cellular and molecular mechanism of MC toxicity and ignore an important aspect of water metabolic abnormalities in animals.The effect of MC on water metabolism in different test animals is unclear.To solve the above problems or answer the above-mentioned questions,this study,based on the previous investigation results,would choose the typical eutrophic lakes in Wuhan and collect dominant cyanobacterial species from water samples in bloom seasons to carry out a series of experiments.These experiments mainly involve four major aspects.Firstly,the characteristics of growth and the toxins of the dominant cyanobacterial species would be figured out.Secondly,the circadian rhythms of growth and psa B,psb D1,rbc L,mcy B,and mcy E gene expression in Microcystis aeruginosa species would be explored.Thirdly,the effects of light combined with different light intensities and periods on the growth and microcystin production of Microcystis sp.would be invesgated.Lastly,abnormal water metabolism between Sprague-Dawley(SD)Rats and Kunming(KM)Mice intoxicated by MC-RR would be compared.Part I Isolation of toxic Microcystis strains and exploration of their characteristics of growth and toxinsObjective: Isolation,cultivation and identification of microcystin-producing strains of Microcystis from water blooms was performed to understand the features of harmful algal blooms in Wuhan lakes and the obtained pure cultures were used for further study on the physiological growth characteristics of Microcystis.The unknown kinetics of growth of the purified and cultured toxic strains,and their toxin-producing capabilities after continuously subculture for a long time would be observed and evaluated separately.Methods: Samples of water blooms were collected from five lakes in the city of Wuhan for strain isolation and MC determination.Strains of Microcystis were isolated by combining the methods of using capillary pipettes to select single colony and spreading the petri dish.The isolated strains were identified by their sequences of the phycocyanin intergenic spacer and the morphological characteristics of the colonies to determine their genera and species.Then three microcystin biosynthesis gene clusters(mcy A,mcy B and mcy D)were measured with PCR for screening the toxic strains,and the high performance liquid chromatography(HPLC)analysis was used to identify the MC analogues in the toxic strains.The growth curves were estimated by cell counts or cellular turbidity measurement and the abilities of the microcystin-production of the toxic strains and the toxigenic duration under the cultural conditions were evaluated by PCR and HPLC methods.Results: MC-RR and MC-LR were detected in the water blooms of the investigated lakes,and MC-RR accounts for a higher proportion.Fifteen Microcystis strains were isolated from the water blooms,which contained five Microcystis ichthyoblabe,two Microcystis novacekii,two Microcystis aeruginosa,one Microcystis flos-aquae and Microcystis wesenbergii,and four Microcystis sp.that cannot be classified into species.Seven toxic strains were identified by PCR and HPLC,in which MC-LR was the only kind of MC detected from the strains of DL2,DL3,ZP2 and ZP3 with reference to three MC standards(MC-RR,MC-YR and MC-LR).The MC-LR and MC-RR were both detected from the strain LH1,MC-RR and a small amount of MC-YR were found in the strain ZP7,while three kinds of MC,MC-RR,MC-YR and MC-LR,were all detected from the strain DL1.By comparison the amount of MC-RR being the major component was much higher than that of the other two kinds of MC in strain DL1.The growth cycle of Microcystis strains ZP2,ZP3,and ZP7 exceeded 60 days.The cell counting results and absorbance values of the cultivated strains had a good consistency in the early growth stage.When the cells entered into platform stage or stationary phase,the changes of absorbance value were relatively lagging behind.After one year's cultivation,the morphology of all the strains changed from larger colony to small colony or single cell,and toxic strains were relatively stable in their toxin-producing ability.Conclusion: MC-RR was dominant among the mixed MC in investigated lakes.Seven microcystin-producing strains of Microcystis were isolated from water blooms in Wuhan lakes,and were stable in the ability of MC production.The toxins produced by these strains varied in levels and types but a general rule were consistent with the patterns found in natural water blooms.The culture cycle of the strains isolated were longer than 60 days in the laboratory,and the absorbance value of cultures in liquid media could reflect the growth of cells in the logarithmic growth phase.The toxin contained in strain DL2 is MC-LR unique within MC congeners and the strain with high toxin production was stable in the culture,so strain DL2 is a suitable object for further study of physiological characteristics in Microcystis and is convenient for MC extraction and purification.Part? A preliminary exploration of the circadian rhythms in Microcystis sp.during the changing of day and night alternationObjective: To explore whether the circadian rhythms in Microcystis sp.is regulated by the endogenous circadian clock or only controlled by the light exposure.Methods: Three test groups and one control group were set for Microcystis aeruginosa FACHB-905 after acclimatization under 12 h:12 h light and dark cycle for exploring the circadian rhythms.The control group was still under the 12 h:12 h cycle.The first test group was a light disturbance group with 19.5 h in light and 4.5 h in dark,which made the cells of FACHB-905 strain to be exposed to light 7.5 h earlier than the control group.The second test group was in continuous illumination and the third test group was in continuous dark.The beginning of the dark phase of the control group was the starting point of the whole experiment and two hours later the sample collection was performed for all the groups,then the sample collection was changed to once every four hours.The optical absorbance of cellular turbidity was measured at 450 nm and 680 nm separately and cells were counted at the end of experiment for evaluating the growth.The expressions of the psa B,psb D1,rbc L,mcy B,and mcy E genes were determined by Real-time PCR for assessing whether they were controlled by the endogenous circadian clock or only by the light.Results: The optical absorbance of culture media and expressions of the psa B,psb D1,and rbc L genes in all groups increased with the light exposure,and declined in the darkness.The expressions of the mcy B and mcy E genes increased after light phase starting,and showed no obvious regularities under the continuous illumination and continuous dark.Conclusion: The circadian rhythms in cell growth and expressions of the psa B,psb D1,rbc L,mcy B,and mcy E genes in Microcystis aeruginosa FACHB-905 were mainly controlled by the light exposure and least by the endogenous circadian clock regulation.Part ? Effects of light combined with different light intensities and periods on the growth and microcystin production of Microcystis sp.Objective: Exploring the effects of light with different light intensities and periods on the growth and microcystin production for Microcystis sp..Methods: A unicellular Microcystis sp.DL2 was used to explore the effects of light on the growth and microcystin production.The strain DL2 was treated with two groups of light conditions keeping total light power as a constant in the groups.Group A: 12.5 ?mol m-2 s-1 with 16 h:8 h light and dark(16 L:8 D)cycle;Group B: 25.0 ?mol m-2 s-1 with 8 h:16 h light and dark(8 L:16 D)cycle.The cell growth and MC production of the strain were compared under the two light conditions.Then,light intensity and light period were split as two variables to evaluate the influence of them on the growth and MC of the strain DL2.(1)Three light intensities of 12.5,25.0,and 31.2 ?mol m-2 s-1 were studied on the cell growth and MC production of the strain DL2.(2)Five light:dark(LD)cycles from 8,10,12,14 to 16 hours light time for twenty-seven days were set to explore the effects of light period on the growth.The effects of light period on MC production were evaluated through MC contents and expression of microcystin biosynthesis genes(mcy B and mcy E)during the fast growth phase and late-log phase under the five LD cycles.MC content was measured and identified by HPLC/MS/MS.The 24 h dynamic changes of mcy B and mcy E transcription levels of DL2 in three LD cycles of cultivation: 9 L:15 D,12 L:12 D,and 15 L:9 D were monitored by Real-time PCR for exploring the mechanism of LD cycle influence.Results: Cell denisties in group A were higher than that in group B,while MC-LR content in group B was higher than that in group A.Elevation of the light intensity from 12.5 ?mol m-2 s-1 to 31.2 ?mol m-2 s-1 could increase cell denisty and MC-LR content for DL2.Cell densities of the cultures increased with the light period extending from 8 L:16 D to 16 L:8 D.However,different LD cycles showed no effects on MC production of strain DL2,no matter on the MC-LR levels or the mcy B and mcy E transcription levels during the early growth phase and the late-log phase.The mcy B and mcy E transcription levels rose transiently at 4 h after the start of light phase under the three different LD cycles in the 24 h monitoring experiment.Conclusion: Although short-term high-light intensity exposure have the same total photons as long-term low-light intensity exposure with regards to physical measurement,their effects on the cell growth and MC production of strain DL2 were not the same.Increase in the light intensity from 12.5 ?mol m-2 s-1 to 31.2 ?mol m-2 s-1 could improve the cell growth and MC-LR content for the strain DL2.Extending the light period from 8 L:16 D to 16 L:8 D could promote the cell growth of the strain DL2,but have no effects on MC production.The rise in transcription levels of mcy B and mcy E genes at 4 h after the start of light phase were transient and were not affected by the light period.Part ? Differences in abnormal water metabolism between SD rats and KM mice intoxicated by microcystin-RRObjective: To explore and compare the effects of MC-RR on water metabolism in SD rats and KM mice.Methods: Firstly,SD rats and KM mice were treated with single dose of MC-RR.Water intake,urine output,and weight gain were recorded,and the changes of liver and kidney were observed including related serological and urinary indices.Four groups of rats were separately given intraperitoneal injections of MC-RR 574.7 ?g/kg,287.3 ?g/kg,143.7 ?g/kg,and physiological saline as control.MC-RR was administered to four groups of mice at 210.0 ?g/kg,105.0 ?g/kg,52.5 ?g/kg,and physiological saline as control.Different doses of dexamethasone were used to block the rat nephrotoxicity in the 574.7 ?g/kg group,and the changes of the positive indexes were monitored.Secondly,a continuous exposure experiment was performed on KM mice to explore the possible impact on water metabolism and related organs.The MC-RR dosages for the four groups were: 140.0 ?g/kg,70.0 ?g/kg,35.0 ?g/kg,and physiological saline as negative control.The mice were given intraperitoneal injection daily for 28 days,and continued to be observed until the 42 nd day.Daily weight,drinking and urine volume were recorded.The urinalysis,serological analysis,and histological examination of kidney and liver were also carried out.Results: In the single dose toxicity test,polydipsia,polyuria,hematuria,and proteinuria were found in group of rats receiving a 574.7 ?g/kg of dose of MC-RR,and could be relieved by dexamethasone.Gradient damage was observed in kidney and liver in rats with gradient MC-RR doses of 574.7,287.3,and 143.7 ?g/kg.No significant water metabolic changes or kidney injuries were observed in mice treated with MC-RR doses of 210.0,105.0,and 52.5 ?g/kg.In the continuous exposure test,in which mice were administrated with 140.0,70.0,and 35.0 ?g/kg MC-RR for 28 days,mice in the 140.0 ?g/kg group presented increasing polydipsia,polyuria,and liver damage.However,no anatomic or histological changes,including related serological and urinary indices,were found in the kidney.The water metabolic dysfunction and liver damage on mice were reversible when administration of the toxin stopped.Conclusion: Abnormal water metabolism can be induced by 574.7 ?g/kg of MC-RR in SD rat through kidney injury.The kidney of SD rat is more sensitive to MC-RR than that of KM mouse.The polydipsia and polyuria in KM mice exposed to 140.0 ?g/kg of MC-RR for 28 days occurred but could not be attributed to kidney damage.