| Microplastics(MPs)are plastic fragments or particles with a particle size less than 5 mm.In recent years,the quantity of MPs in marine environment is increasing,and the impact of MPs on marine organisms has attracted extensive attention.MPs often affect organisms together with other pollutants,so the combined effect of microplastics and other pollutants becomes a hot area of research.Nereis are benthic invertebrates living in marine-terrestrial interlaced zone.It has been found that some worms have strong tolerance to pollutants and can be used as an important organism for marine pollution remediation.However,there are few studies on the toxicity effects of the combination of MPs and PAHs on Nereis.In this study,we chose Perinereis aibuhitensis as experimental animals and polyethyleneterephthalate(PET)and benzo[a]pyrene(B[a]P)as exposure pollutants.Based on previous study in our laboratory,100?g/L microplastic group,5?g/L B[a]P group,100?g/L microplastic +5?g/L B[a]P group and blank control group were set for exposure experiment.Firstly,the changes of enzyme activity was detected,then the different expression genes was selected through tanscriptome sequencing,finally the gene expression of key genes under single and combined exposure of MPs and B[a]P was analyzed by real-time quantitative PCR.The main results are as follows:1.The changes of SOD,CAT and GST activities and LPO content in different treatment groups at 1,4,7 and 10 days were determined by using enzyme activity kit.It indicated that the SOD,CAT and GST activities and LPO content in P.aibuhitensis under exposure was induced,and the SOD,CAT and GST activities and LPO content in combined induction group was much higher than other two treatments in day 1.The SOD and CAT activity in MP group decreased from day1 to day4,then increased from day7.The GST activity and LPO content also decreased from day1 to day4,and increased at day7 then decreased at day 10.The SOD and CAT activity in B(a)P group decreased from day 1 to day 7,and then increased.GST activity in B(a)P group decreased from day 1 to day 4,and then increased.But LPO content in B(a)P group changed smoothly.The SOD,CAT and GST activities in combined group reached the maximum in day 1,then decreasd finally increased at day 10.2.The tanscriptome was constructed using the body wall of worms in different exposure groups in day 1,and the Differential expressed genes was analyzed.The results showed that there were 7506 differentially expressed genes in MPvs Blank which is the highest,including 3710up-regulated genes and 3796 down-regulated genes.And there were 4999 differentially expressed genes in MIXvs Blank which is the lowest,including 2995 up-regulated genes and2004 down-regulated genes.KEGG analysis showed that genes in propanoate and nitrogen metabolism changed a lot under MPs and B(a)P single and combined exposure.The expression genes related to dexofication metabolism including cytochrome P450 and ABC transportersalso changed under B(a)P single and combined exposure.Furthermore,the expression of genes related to lysosome regulatory pathway was only changed in combined group.Genes includinng cytochrom?B561,UDP-glucuronosyltransferase 1A7 C,ABC transporter B family member 1,glutathione peroxidase 6,heat shock protein beta-1,mitochondrial import receptor subunit TOMM70,ferritin,lysosomal aspartic protease was selected for vertification,and the results showed that except for glutathione peroxidase 6 all of those gene expression was similar with the results in transcriptome.3.The expression characteristics of cytochrom?B561,UDP-glucuronosyltransferase 1A7 C,ABC transporter B family member 1,glutathione peroxidase 6 and heat shock protein beta-1 in different groups were analyzed by fluorescence real-time quantitative PCR.The results showed that gene exression of those five genes was induced under exposure.The gene expression of glutathione peroxidase 6 in all treatmens was induced,and it means that pollutant exposure causes oxidative stress to P.aibuhitensis.The expression of cytochrom?B561 in combined exposure group was induced gradually and higher than that in B(a)P group.The expression of UDP-glucuronosyltransferase 1A7 C in B(a)P single group was highest in day1,then the expression in combined group increased gradually,and higher than that in B(a)P group at day7 and 10.The expression fo ABC transporter B family member 1 in B(a)P and MP group reached the maximum at day 4,but the expression in combined group increased and higher than that in single group in day 7 and day 10.The gene expression of glutathione peroxidase 6in combined exposure group was higher than that in single exporsure group except at day 1and day 10.the gene expression of heat shock protein beta-1 reached the maximum in MP group in day 4,then decreased.However,the expression of heat shock protein beta-1 in B(a)P single and combined exposure groups reached the maximum in day10 and day 7,respectively,and the expression in combined exposure group was higher than other groups in day 4 and 7.All of those results indicates that There is a synergistic effect on P.aibuhitensis gene expression under microplastics and B[a]P combined exposure... |
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