| Strobilurin fungicides(SFs)are an important agricultural fungicide and the best-selling fungicides.SFs are a kind of mitochondrial respiration inhibitor,which inhibits the mitochondrial respiration of target fungi by transferring electrons between cytochrome b and c1,thereby inhibiting the synthesis of adenosine triphosphate(ATP)and causing oxidative stress damage.There have been studies that have detected different concentrations in various environmental samples including soil samples.Therefore,it is particularly essential to study the toxic effects and mechanisms of such fungicides on non-target organisms.Fluoxastrobin(FLUO),the typical SFs,is widely used in the treatment of stems and leaves,and can effectively prevent fungal diseases of vegetables,potatoes and etc.With the use of FLUO,it will accumulate in the soil environment with the degradation half-life of 16?119 and affect the stability of the ecosystem.Earthworms are an important part of terrestrial ecosystems and can be used to assess the risks of pollutants to soil organisms.The Organization for Economic Cooperation and Development(OECD)defines Eisenia fetida as the soil model animal.In recent years,studies on the toxicity of SFs to non-target organisms in different environments have attracted widespread attention.However,there are relatively few studies on FLUO,and the study on the toxic effect of Eisenia fetida is almost blank,only its study in artificial soil.Using artificial soil for toxicological tests can better control the soil conditions and ensure that the toxicological results are not affected by the soil conditions so that the toxicity of different pollutants can be compared.However,under realistic conditions,pollutants enter different types of soil through various channels,and different physical and chemical properties of soil will affect the toxicity of pollutants on earthworms.This led to a question,that is,whether the toxicity of FLUO on Eisenia fetida in artificial soil can accurately represent the toxicity that it may cause in natural soil?Therefore,this study selected the real field soil(fluvo-aquic soil,red clay,and black soil)in China,using reactive oxygen species(ROS),the activity of superoxide dismutase(SOD),catalase(CAT),and glutathione-S-transferase(GST),and the contents of malonaldehyde(MDA)and 8-hydroxy deoxyguanosine(8-OHdG)as the endpoints to evaluate the toxic effects of FLUO(control group(NF)and 0.1 mg kg-1(LF),1.0 mg kg-1(MF),2.5 mg kg-1(HF))on Eisenia fetida and its mechanism after 28-day exposure.To more clearly compare the toxicity of FLUO to Eisenia fetida in different soil types,we selected the ROS contents,MD A contents,and three enzyme activities of the HF treatment after 4-week exposure to calculate the integrated biomarker response(IBR v2)index.This topic also uses transcriptomics(RNA-seq)and proteomics(iTRAQ)to study the different changes of the gene expression and protein expression to explore the toxic mechanism of FLUO to Eisenia fetida in fluvo-aquic soil.The following results and conclusions can be obtained through this topic.1)Fluoxastrobin can induce Eisenia fetida to produce oxidative stress and DNA damage in different types of soils.In different soils,FLUO can increase the ROS contents in earthworms,and it gradually increased with the increase of exposure concentration.It has a certain dose-effect relationship and also has an impact on the activity of antioxidant enzymes(SOD and CAT)in earthworms.FLUO exposure can also cause lipid peroxidation and DNA damage in earthworms,which is specifically manifested by the increase in MDA contents and 8-OHdG contents.2)According to the IBR index,it is concluded that FLUO has different toxicity performance to Eisenia fetida in different types of soil:red clay>fluvo-aquic soil>black soil>artificial soil;therefore,we believe that the toxicological results of FLUO in artificial soil cannot accurately assess its true toxicity in the environment.Besides,SFs.can have varying degrees of toxic effects on soil animals Eisenia fetida in different soils.3)The present study compared the differential expression of Eisenia fetida before and after FLUO treatment at the transcriptome level.Besides,it also analyzed the differentially expressed genes(DEGs),their functions,and metabolic pathways.(a)After treatment with different concentrations of FLUO,the expression of genes in Eisenia fetida cells at the transcriptional level varied:there are 225 DEGs in NF and LF(128 genes are up-regulated and 97 genes are down-regulated compared with NF);352 DEGs in NF and MF(224 genes are up-regulated and 128 genes are down-regulated compared with NF);and 296 DEGs in NF and HF(160 genes are up-regulated and 136 are down-regulated genes compared to NF).(b)27 genes that significantly changed under different concentrations of FLUO treatment were screened.These genes are involved in protein folding,classification and degradation,signal transduction,immunity,cell growth,and other related biological pathways.FLUO serves as a kind of SFs,which caused certain stress to the normal basic physiological activities of Eisenia fetida.(c)Eisenia fetida has the stress response to different concentrations of FLUO:low concentration(0.1 mg kg-1)of FLUO mainly affects the gene function and kinase activity related to the extracellular region;medium concentration(1.0 mg kg-1)of FLUO induces many different functional gene-encoded proteins in Eisenia fetida cells to produce a stress response.Significant changes have occurred in the pathways of basic substance metabolism;and under the condition of high concentration(2.5 mg kg-1)of FLUO,the most gene functions and pathways in Eisenia fetida are significantly inhibited,and immune-related functional genes and pathways have its effects.In general,Eisenia Fetida cells in each treatment group showed apoptosis,and the degree of apoptosis increased with the increase of FLUO concentration.4)The present study compared the differential expression of Eisenia fetida before and after FLUO treatment at the proteome level.Besides,it also analyzed the differentially expressed proteins(DEPs),their functions,and metabolic pathways.(a)After treatment with different concentrations of FLUO,the protein expression levels in Eisenia fetida cells varied:there are 261 DEPs in NF and LF(74 proteins are up-regulated and 187 proteins are down-regulated compared with NF);383 DEPs in NF and MF(137 proteins are up-regulated and 246 proteins are down-regulated compared with NF);330 DEPs in NF and HF(108 proteins are up-regulated and 222 proteins are down-regulated compared with NF).(b)59 proteins that significantly changed under different concentrations of FLUO treatment were screened.These proteins have the apoptosis-related functions and are involved in signal molecules and interaction,cell growth and death,signal transduction,immune system,and other related biological pathways.FLUO affected kinase activity,inhibited ATP synthesis to cause oxidative stress,induce cell apoptosis in Eisenia fetida in each treatment group,and the degree of apoptosis increased as the concentration of FLUO increases.(c)There was no common significant enrichment pathway in the three treatment groups of NF-vs-LF,NF-vs-MF,and NF-vs-HF.Eisenia fetida has the stress response to different concentrations of FLUO:low concentration(0.1 mg kg-1)of FLUO has no significant effects on the structural molecule activity;medium concentration(1.0 mg kg-1)of FLUO induces oxidative stress in Eisenia fetida and immune-related functional proteins and pathways began to take effect;under the condition of high concentration(2.5 mg kg-1)of FLUO,the most protein functions and pathways in Eisenia fetida are significantly inhibited,and induce the immune-related function.The present study systematically studied the toxic effects of FLUO on Eisenia fetida in different soils and compared its toxicity differences in different soils.The study of Eisenia fetida transcriptomics and proteomics explored the toxicity mechanism of FLUO to Eisenia fetida.The present study has clarified more toxicological data about SFs and provided a scientific basis for evaluating the toxicity mechanism of FLUO and the other SFs to earthworms. |
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