Biochemical And Molecular Response Of Dugesia Japonica To Glyphosate Stress

With the rapid development of modern agriculture,the application of pesticides is becoming more and more extensive.As the worldwide top-selling herbicide,glyphosate is widely distributed in the natural environment,and its impact on the ecological environment and human health has been increasingly concerned.It has been reported that glyphosate has toxic effects not only on aquatic animals such as fishes and soil animals such as earthworms,but also on immune system,endocrine system and reproductive system of rats and other mammals as well as human beings.However,toxicological study of glyphosate on planarians has rarely been reported so far.Planarians distribute widely in springs and streams all over the world and is an important member of the freshwater ecosystem.Since planarians are highly sensitive to toxic factors in the environment,sublethal doses of pesticides and heavy metals can cause the damage of them from DNA molecules to individual levels.Dugesia japonica is a freshwater planarian species widely distributed in East Asia.It is an important model species for planarianoloy study in the world,and has gradually become a model animal for toxicological studies in recent years.In this study,the acute toxicity of glyphosate to D.japonica was studied by acute toxicity test.Based on this,the mRNA expressions of important biomarker genes?SODs,CAT,GPX,Hsp70,Hsp90 and CYP450?and the enzyme activity of main antioxidant enzymes in planrians upon glyphosate exposure were explored by quantitative real-time PCR?qPCR?and biochemical analysis.It will provide valuable information for ecological safety and environmental risk evaluation and theoretical guidance for reasonable application of herbicide glyphosate.The main conclusions we have drawn are as follows:1.The full-length cDNA of CAT in D.japonica?DjCAT?is 1677 bp,including an open reading frame?ORF?of 1461 bp encoding a polypeptide of 486 amino acids.DjCAT has no signal peptide and is a peroxisomal glycoprotein with two glycosylation sites.It is speculated that DjCAT has a symmetrical homologous tetrameric structure.DjCAT contains two CAT family signature sequences of a proximal active site signature(⁴⁰FDRERIPERVHAKGGA⁵⁷)and a heme-ligand signature motif(³³⁰RLFSYRDTQ³³⁸)and three catalytic amino acid residues of His51,Asn124 and Tyr334.The highest identity is presented at79%with planarian Schmidtea mediterranea.2.The 50%lethal concentration(LC₅₀)?96 h?of glyphosate on D.japonica is 128 mg/L,and its 95%confidence limit?95%CI?is 108-199 mg/L.The LC₅₀ of glyphosate on D.japonica is higher than that of some other organisms such as Macrobrachium nipponensis and Xenopus lavis.According to the international toxicity classification criterion,glyphosate is slightly toxic to D.japonica.3.The relative mRNA expressions of main antioxidant enzyme genes?CuZnSOD,MnSOD,CAT and GPX?and important biomarker genes?Hsp70,Hsp90 and CYP450?were altered in D.japonica exposed in glyphosate of different concentrations for different time.This indicates that D.japonica has shown molecular response to glyphosate.4.The activity of the main antioxidant enzymes?SOD,CAT and GPX?in D.japonica treated with glyphosate of different concentrations for different time were changed,and the level of T-AOC and the content of MDA were also varied.This suggests that D.japonica has shown biochemical reponse to glyphosate.Several antioxidant enzymes in the planarian can work together to effectively remove ROS and maintain the balance of oxidation/antioxidation.