| Rotifera species are the key component of primary consumers in the water ecological systems and affect the composition and number of phytoplankton through feeding control.Changes of the number of rotifers lead to output fluctuation of high trophic levels of fish,shrimp,and crab.Therefore,rotifers are usually considered a ligament that connects the primary producer and the high trophic levels.The growth conditions and population changes of rotifers play a key role in the dynamics of aquatic ecosystems.Thus,rotifers are often defined as an "ecological interactor".Brachionus calyciflorus is used as a model animal in an ecotoxicology study because of their wide distribution,small size,short life cycle,ease of culture,and sensitivity to toxins.Eutrophication causes blue green algae to become the dominant species of the phytoplankton community and even causes an outbreak of cyanobacterial bloom.This phenomenon is particularly significant in the summer high-temperature stage.Microcystis aeruginosa is the most studied and is one of the most harmful cyanobacterial bloom.Microcystis releases toxins,lacks of key nutrients,and gather to form groups,leading to the inhibition of the growth and reproduction of rotifers.Moreover,in an environment of long-term coexistence with Microcystis,rotifers can graze on Microcystis,inhibit the excessive multiplication of Microcystis,and improve the resistance to microcystins.In the interaction between Microcystis and zooplankton,rotifers change the physiological and biochemical characteristics and the life history to maximize their-fitness in response to Microcystis and its derivatives.What are the ecological effects of water bloom Microcystis and its derivatives?What are the changes of the antioxidant system under stress of Microcystis and its derivatives?What is the molecular mechanism of rotifers in response to microcystin-producing M.aeruginosa.This study addresses these scientific problems using classic molecular ecology and biology methods.This study focuses on the ecological characteristics and molecular mechanism of rotifer tolerance to water bloom Microcystis and its derivatives.1.Effects of microcystin-producing and microcystin-free M.aeruginosa on life history and grazing intensity of B.calyciflorus.Population and individual experiments were conducted with the same proportional volumes of Chlorella and Microcystis for given food densities.Life-table parameters,life-history traits,and grazing intensity of B.calyciflorus were evaluated after they had fed on microcystin-producing and microcystin-free Microcystis,i.e.,alone and combined with an edible alga(Chlorella pyrenoidosa),at concentrations of 1 × 105,1 × 106,and 1 × l07 cells mL-1.Results showed that the interactive effects of food density and type appeared to be synergistic on generation time(T),net reproduction rate(R0),body length,swimming speed,and reproduction time(p<0.05).By contrast,these effects appeared to be antagonistic on intrinsic growth rate(rm),finite rate of increase(?),time to first brood,post-reproductive time,and total offspring per female(p>0.05).The grazing rate of rotifers decreased with grazing time.Although the toxins released after grazing on M.aeruginosa had negative effects on rotifer growth and reproduction,B.calyciflorus changed its life history traits and grazing intensity in response to eutrophic conditions.2.Effects of microcystin-producing and microcystin-free M.aeruginosa on the enzyme activity and nutrient content of the rotifer B.calyciflorus.The freshwater cyanobacterium M.aeruginosa produces microcystins,which are compounds toxic to rotifers.This study evaluated the effects of M.aeruginosa on the enzyme activity and nutrient content of the rotifer B.calyciflorus.The rotifers were fed with C.pyrenoidosa,Scenedesmus obliquus,microcystin-producing and microcystin-free M.aeruginosa alone,and mixtures of green algae combined with toxic and nontoxic cyanobacteria.The activities of amylase,pepsase,trypsin,cellulase,superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GPx)were assessed after rotifer exposure to an environmental stressor.The nutrients analyzed included glycogen,protein,and triglyceride(TG).Single cyanobacteria and mixtures combined with toxic M.aeruginosa inhibited SOD activity.CAT and GPx activities significantly increased in rotifers fed with the mixture of Chlorella and toxic cyanobacteria.The activity of digestive enzymes increased compared with the Chlorella group in single and mixed diets.Glycogen and protein decreased in Microcystis mixtures,whereas TG content increased.The grazing rate(G)of rotifers decreased with grazing time.A high G value was observed with green algae in every treatment group.Although the toxins released after grazing on Microcystis affected rotifer enzyme activity and nutrient content,B.calyciflorus changed its physiological performance and grazing intensity with food type in response to eutrophic water environment.3.Interactive effects of microcystin and ammonia on the reproductive performance and phenotypic traits of the rotifer B.calyciflorus.Elevated microcystin-LR(MC-LR)and ammonia(NH3-N)concentrations co-occur during the degradation of Microcystis blooms and are toxic to aquatic organisms.The freshwater rotifer B.calyciflor was exposed to mixtures of MC-LR(0,10,30,and 100 ?g L-1)and NH3-N(0,270,and 540 ?g L-1)to assess the combined effects of the two toxicants on reproductive performance and phenotype traits.Single solutions of MC-LR(100 ?g L-1)and NH3-N(540 ?g L-1)had negative effects on rotifer reproductive timing and fecundity.The pre-reproductive and post-reproductive periods fluctuated with the MC-LR and NH3-N concentrations,whereas the reproductive period and total offspring per female were reduced in mixtures of MC-LR and NH3-N(p<0.05).The grazing rate of rotifers decreased with grazing time and concentrations of the two toxicants(p<0.001).MC-LR in combination with NH3-N had negative effects on swimming speed and body length but positively stimulated posterolateral spine development(p<0.001).MC-LR and NH3-N had synergetic interactive effects on pre-reproductive period,reproductive period,total offspring per female,grazing rate,swimming speed,and body length(p<0.05).By contrast,these effects were antagonistic on post-reproductive period and posterolateral spine length(p>0.05).These results indicate that MC-LR and NH3-N act synergistically and antagonistically in causing toxicity to B.calyciflorus regarding reproductive performance and the formation of defensive phenotypes.4.Effects of microcystin and ammonia on the life table parameters and glutathione(GSH)-related enzymes of the rotifer B.calyciflorus.In this study,B.calyciflorus was exposed to solutions with different combined concentrations of MC-LR(0,10,50,100,and 200 ?g L-1)and NH3-N(0,740,and 1,580 ?g L-1)to assess the combined effects of MC-LR and NH3-N on life-cycle parameters and oxidative stress.Single solutions of MC-LR 200 ?g L-1 and NH3-N 1,580 ?g L-1 and mixtures of the two toxicants were observed to be toxic to rotifers in terms of net reproduction rate(R0),intrinsic growth rate(r.),activities of GSH,GPx,and glutathione S-transferase(GST),and phospholipid hydroperoxide glutathione peroxidase(PHGPx)expression.MC-LR combined with NH3-N decreased the generation time(T),Na+/Kt-ATPase activity,and glutathione reductase(GR)activity but increased the reactive oxygen species(ROS)levels(p<0.05).ROS levels were negatively correlated with GSH-related enzyme activities(p<0.01).MC-LR and NH3-N exhibited interactive effects on R0,rm,ROS levels,activities of GSH,GR,and GST,and PHGPx expression(p<0.05).By contrast,these effects were antagonistic on T,Na+/K+-ATPase activity,and GPx activity(p>0.05).The results further showed that cyanobacterial metabolites acted synergistically and antagonistically to cause toxicity to B.calyciflorus.The life-cycle parameters of rotifers were associated with the effects on the GSH-related defense system in response to MC-LR and NH3-N.5.Effects of microcystin and nitrite on the lifespan and heat shock responses of the rotifer B.calyciflorus at different temperatures.Toxicants such as MC-LR and nitrite(NO2-N)that are released during the degradation of cyanobacterial blooms have adverse effects on zooplankton.In this study,the rotifer B.calyciflorus was exposed to solutions with different combined concentrations of MC-LR(0,10,30,and 100 ?g L-1)and NO2-N(0,1,3,and 5 mg L-1)to assess the combined effects of these toxicants on the lifespan and responses of heat shock proteins(hsps)at 20 ?,25 ?,and 30 ?.Rotifer lifespan was shortened in single solutions of MC-LR 100 ?g L-1 and NO2-N 5 mg L-1.The expressions of hspsfluctuated with toxicant concentrations,whereas the ROS levels increased in every treatment at each temperature(p<0.05).Oxidative stress induced by MC-LR and NO2-N negatively affected the lifespan and hsps expressions at 30 ?.MC-LR and NO2-N had synergic effects on the lifespan and hsps expressions at 20 ? and 25 ?(p<0.05)but had antagonistic effects on the expressions of hsp40 and hsp60 at 30 0C(p>0.05).Temperature,MC-LR,and NO2-N had interactive effects on the lifespan,ROS levels,and hsps expressions(p<0.05).Cyanobacterial metabolites cause toxicity to rotifers,and the expressions of hsps are useful biomarkers of exposure to MC-LR and NO2-N.6.Combined effects of microcystin and nitrite on the growth,lipid peroxidation,and antioxidant responses of the freshwater rotifer B.calyciflorus.MC-LR and NO2-N affect the growth of aquatic organisms.The freshwater rotifer B.calyciflorus was exposed to solutions with different combined concentrations of MC-LR(0,10,50,100,and 200 ėg L-1)and NO2-N(0,2,4,6,and 8 mg L-1)to assess the combined effects of MC-LR and NO2-N on life-cycle paraneters and oxidative stress.Single solutions of MC-LR 200 ėg L-1 and NO2-N 8 mg L-1 were toxic to rotifers.MC-LR combined with NO2-N decreased population growth rate(r),survival,and reproduction but increased ROS,malondialdehyde(MDA),and GSH contents(p<0.01).SOD and CAT activities and mRNA expression levels of MnSOD,CuZnSOD,and CAT significantly decreased under high concentrations of MC-LR or NO2-N(p<0.05).ROS levels had negative correlations with antioxidant enzyme activities and expression levels of antioxidant genes(p<0.01).MC-LR and NO2-N had interactive effects on r,reproduction,ROS levels,MDA content,SOD activity,and expression levels of MnSOD and CAT(p<0.05).By contrast,these effects were antagonistic on survival,CAT activity,GSH content,and expression level of CuZnSOD(p>0.05).The MnSOD gene had the highest expression under stress of MC-LR and NO2-N,and the MnSOD in vitro prokaryotic expression of purified protein had the highest activity in vitro at 30 ?,pH 9.0,and 2%SDS mutagen induction,indicating that high-temperature and highly alkaline environments have an influence on the antioxidant system of rotifers.These results showed that MC-LR and NO2-N cause toxicity to B.calyciflorus.ROS-mediated toxicity was considered the mechanism by which MC-LR and NO2-N induce damage.7.Molecular mechanism of B.calyciflorus in response to microcystin-producing M.aeruginosa at the transcriptome levels.This study first analyzed B.calycriflorus in response to microcystin-producing M.aeruginosausing the population growth rate indicator(r)and determined that the r value significantly decreased with rotifers grazing on 100%toxic M.aeruginosa(p<0.05),indicating that microcystin-producing M.aeruginosa was harmful to rotifers.Then,this experiment combined with modern high-throughput RNA sequencing technology,compared the differential genes between transcriptomes of rotifers fed with 100%toxic Microcystis and with the ideal food of 100%Chlorella.For the first time,data showed that the genetic change of rotifer in response to toxic Microcystis can be mainly categorized under three broad headings,i.e.,biological process,cellular component,and molecular function.These results showed that the ecological phenomenon of the reduction of B.calyciflorus population was related to the mostsignificant enrichment of the cell cycle,spliceosome,amino sugar and nucleotide sugar metabolism pathways,RNA metabolic process and apoptosis,affecting the enzyme activities in the antioxidant system and the expression of antioxidant proteins. |
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