| Plastics have been widely used in various fields,such as agriculture,industry,construction,and daily life.Though plastics industry has brought great convenience to people,it has also caused inevitable harm to the environment.Plastics in the environment gradually degrade into microplastics(between 1 and 5000 ?m)and nanoplastics(less than 1 ?m)under the long-term impact by biological,physical,and chemical effects.While microplastics has received much attention,less research has been conducted on nanoplastics,which are smaller than microplastics.Smaller particle size compounds often have unique properties that may cause more serious effects on organisms.Therefore,in the present study,Daphnia pulex and polystyrene(PS)nanoplastics(a commonly used plastic polymer)were selected to systematically study the biological effects of nanoplastics and their mechanisms from different levels,such as molecular,individual,and population.We firstly elucidated the effects of nanoplastics on the ingestion,growth and reproduction of Daphnia pulex;then verified that nanoplastics reduced the feeding of D.pulex and affected the energy-related pathways;then investigated the possible mechanisms of short-term and long-term nanoplastic exposure using transcriptomics and proteomics,respectively,and used related methods to preliminarily verify the molecular mechanisms of the biological effects of nanoplastics.Finally,the effect of environmental predicted concentration of nanoplastics on D.pulex was revealed to be a generational disturbance.The main results were as follows:1 Preliminary study on the effects of nanoplastics on D.pulexTo confirm the effect of nanoplastics in D.pulex,fluorecent nanoplastics was used to determine the ingest of nanoplastics;and then an acute toxicity test was performed for 48 h LC50.Based on LC50 result,a series of nanoplastics concentration(0,0.1,0.5,1,and 2 mg/L)were set and a 21-day chronic toxicity test was conducted.Growth and reproduction parameter and expression of stress defense genes were determined.The results revealed that the digestive of D.pulex were strongly fluorescent after exposure to the nanoplastics for 24 h.The 48 h LC50 of nanoplastics was 76.69 mg/L.Dose-and time-dependent relationships were observed for body length,and the time to eggs and clutch were significantly prolonged.The reproduction and r were decreased.With increasing nanoplastics concentration,antioxidant genes expression was first increased and then inhibited.The gene expressions of heat shock proteins were induced.These results indicated that nanoplastics can be ingested by D.pulex and affect its growth and reproduction.To further confirm the effect of nanoplastics on D.pulex,this study continued to determine the response of different age groups(1-,4-,7-,14-,and 21-d-old)to nanoplastics.We determined LC50 and the expression of stress defense and energy related gene.Except 21-d-old,significant differences were observed in LC50 for age groups(4-,7-,and 14-d-old)in comparison to that for 1-d-old.Nanoplastics affect expression of stress defense and energy related gene in all age groups compared to the control group,while not in the same way for each.Above results suggest that nanoplastics has an effect on D.pulex.2 Effect of nanoplastics on feeding and AMPK-mTOR pathway of D.pulexAs nanoplastics were mainly enriched in digestive,this study speculated that it may affect the feeding of organism,which was similar to large plastic particles.Thus,we further examined the effects of nanoplastics on the gene and protein expression of AMPK-mTOR pathway of D.pulex.The result indicated that,the full length of AMPKa,? and y genes of D.pulex were cloned.Meanwhile,nanoplastics significantly affected the gene and protein expression of AMPK-mTOR pathway.It suggested that nanoplastics enters the digestive tract of D.pulex and may leads to a decrease in food intake,subsequent affects the AMPK-mTOR pathway,and finally affects the organism.3 Transcriptomic and proteomic study of actual and chronic nanoplastic exposure on D.pulexTo detect the molecular mechanisms of nanoplastics on Daphnia,transcriptomic and proteomic analyses were performed for actual and chronic exposure respectively.A total of 208 differentially expressed genes(107 up-regulated and 101 down-regulated)were identified in the transcriptome.The gene functions and pathways for oxidative stress,immune defense,and glycometabolism were identified.A total of 327 differentially expressed proteins were detected by proteome and we proposed an adverse outcome pathway for nanoplastics,including reactive oxygen species production and oxidative stress as the molecular initiating event(MIE);followed by changes in specific signaling pathways(Jak-STAT,mTOR,and FoxO)and in the metabolism of glutathione,protein,lipids,and molting proteins;with a final result of growth inhibition and decrease in reproductive output.Oxidative stress is one of the common affect pathways between transcriptome and proteome.4 Molecular mechanisms of oxidative stress and related pathways in response to nanoplasticsBased on the results of transcriptome and proteome,we centered on the oxidative stress and related pathways.We determined the content of reactive oxygen species(ROS),gene and protein expression in the MAPK-HIF-1/NF?B pathway,and antioxidant gene expressions and enzyme activities in D.pulex exposed to PS nanoplastics.Moreover,extracellular signal-regulated kinases(ERK),GCLC(glutamate cysteine ligase),and HO1(heme oxygenase 1)gene were cloned in D.pulex.The results showed that nanoplastics caused the overproduction of ROS along with dose-dependent effects.Low nanoplastics concentrations(0.1 and/or 0.5 mg/L)significantly increased the expressions of genes of the MAPK-HIF-1 pathway,and the activity of glutathione-S-transferase.As the nanoplastics concentration increased,these indicators were significantly suppressed.The protein expression ratio of ERK,JNK,AKT,HIFla,and NFKBp65(nuclear transcription factor-KB p65)as well as the phosphorylation of ERK and NF?Bp65 were increased in a dose-dependent manner.It suggested that nanoplastics might cause the overproduction of ROS and activate the downstream pathway,resulting in inhibited growth and reproduction.5 Effect of nanoplastics on the D.pulex glutathione s-transferase gene familyGST is the main component of oxidative stress and glutathione metabolism which was affected by nanoplastics.To further confirm the role of GST in nanoplastics exposure,the GSTs1,GSTs2,GSTm1,and GSTm2 genes of D.pulex were cloned.Subsequently,35 GST gene families were identified.These can be divided into 8 subfamilies:sigma,delta/epsilon,mu,zeta,theta,omega,kappa,and microsomal,which included 10,7,7,1,1,2,2,and 5 genes,respectively.The same families often share the same structural domains and there are tandem duplications on chromosomes.The results of expression profiles under nanoplastics exposure showed that GST genes had their own expression characteristics in response to environmental stresses.6 Effect of nanoplastics at predicted environmental concentration on D.pulex after exposure through multiple generationsUsing the biomarkers obtained from above results,this study further investigated the effect of nanoplastics at predicted environmental concentration on D.pulex after exposure through multiple generations.D.pulex was exposed to a predicted environmental nanoplastics concentration(1 ?g/L)over three 21-day generations.Chronic exposure did not affect the survival or body length of D.pulex,while the growth rate and reproduction were affected in the F2 generation.Molecular responses suggested that environmental nanoplastics concentrations can modulate the response of antioxidant defenses,cellular stress,biotransformation,development,and energy related gene in the F0-F1 generations,which resulted in inhibitory effects on antioxidant responses in F2 individuals.Some recovery was observed in the recovery group,but reproduction and stress defenses were significantly induced.It suggested that nanoplastics have potent long-term toxic effects on D.pulex.In conclusion,nanoplastics inhibited growth,del aid reproduction and reduced production of D.pulex.Its reason may be related with the reduced feeding rate,which further affect energy metabolism.It may also be related with the excessive increase of ROS,which affects the downstream pathways and causes oxidative stress.Nanoplastics have potent long-term effects on D.pulex even under the environmental predicted concentration. |
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