Response Of Spodoptera Litura To Zn Stress And And Its Molecular Mechanism

Zinc（Zn）is one of the most important pollutants.When the content of Zn in agricultural soil is more than 300 mg·kg^-1,there is a pollution risk to agricultural products.Herbivorous insects play an important role in the accumulation and transmission of Zn,which are also affected by Zn stress.However,under low intensity or long-time stress of heavy metal,herbivorous insects have adaptability to heavy metal stress,and even improve their tolerance to other stresses（e.g.high temperature,pesticides）.In this paper,herbivorous insect S.litura was used as investigated object.The larvae of S.litura were treated with different concentrations of Zn(0,150,300,450,600 and 750 mg·kg^-1)in artificial diet.Effects of Zn stress on growth,development and food utilization of S.litura were investigated,and Zn ingestion,excretion and accumulation in S.litura were analyzed,in order to calculate the tolerance threshold of S.litura larvae to Zn stress.The molecular mechanism of the response of S.litura to Zn stress was analyzed by transcriptome sequencing,m RNA digital gene expression profile（DGE）and quantitative proteomics（i TRAQ）.After exposed to Zn stress for 96 h,the response of S.litura to high temperature and pesticides（beta cypermethrin,CPM;chlorpyrifos,CPF）was analyzed,in order to reveal ecological consequences of pest adaptability to Zn stress.The main results were as follows:1.The response of S.litura to Zn stress varied with Zn concentration:the adult weight significantly increased under 150 mg·kg^-1Zn treatment;under≥600 mg·kg^-1Zn treatment,the survival rate,pupation rate and emergence rate of larvae significantly reduced;under≥450 mg·kg^-1Zn treatment,the development period of larvae and pupae prolonged while the adult longevity shortened;under≥300 mg·kg^-1Zn treatment,the body weight of the6^thinstar larvae,male and female pupae and female adults decreased significantly.Hence,the tolerance threshold of S.litura to Zn stress was close to 450 mg·kg^-1Zn.2.Under lower than 450 mg·kg^-1Zn treatment,the approximate digestibility（AD）,relative consumption rate（RCR）,Zn absorption rate and excretion rate of the larvae significantly increased.However,under higher than 450 mg·kg^-1Zn treatment,relative consumption rate（RGR）and Zn absorption rate and excretion rate of the larvae significantly decreased.Hence,within the tolerance threshold of S.litura to Zn stress(450mg·kg^-1),Zn accumulation in larvae could be regulated by changes in the ingestion and excretion of Zn.Moreover,150 mg·kg^-1Zn treatment can promote the growth and development of S.litura,while 600 mg·kg^-1Zn treatment significantly inhibited the growth and development of S.litura,both Zn treatments were used in the following experiments.3.After exposed to 150 mg·kg^-1Zn,there were 100 DEGs（27 up-regulated and 73down-regulated genes,respectively）:Unigene9675（CCE016a）that involves in hormone synthesis and fat digestion and absorption pathway was up-regulated;CL1483.Contig1（related to amylase）and Unigene28281（related to protease）that involves in carbohydrate metabolism and protein digestion and absorption were up-regulated.The expression of Unigene22692（Hsp70）,involving in drug metabolism,was down-regulated.After exposed to 600 mg·kg^-1Zn,there were 447 DEGs（215 up-regulated and 232 down-regulated genes,respectively）:Unigene14445（CYP6AB14）that involves in fat digestion and hormone biosynthesis was up-regulated;Unigene6753 that involves in carbohydrate and fat digestion and absorption was down-regulated;and Unigene22692（HSP70）and Unigene 33678（UGT46A4）,involving in drug metabolism of other enzymes,was down-regulated.After exposed to 150 mg·kg^-1Zn,there were 112 DEPs（up-regulated and down-regulated proteins were 39 and 73,respectively）:the content of CYP321B1（CL360.Contig1）involving in hormone synthesis,drug metabolism and fat digestion and absorption increased;the content of CL317.Contig2 and Unigene 9676（CCE016a）involving in steroids synthesis and fat digestion and absorption increased;the protein of s Hsp19.3（Unigene 28432）and s HSP（Unigene 29830）involving in hormone biosynthesis were down-regulated.After exposed to 600 mg·kg^-1Zn,there were 73 DEPs（32 up-regulated and 41 down-regulated,respectively）,including CYP6B50（CL3369.Contig2,CL557.Contig1）,GST1（Unigene 28619）,GSTO2（Unigene 6526）,GSTe2（Unigene 719）,GSTs1（Unigene 28047）and GSTs3（Unigene 8124）,which involved in drug metabolism and hormone synthesis.In the metabolic pathway related to function,the differentially expressed genes or proteins（CL637.contig2,Unigene28281,Unigene9675,Unigene29369,Unigene14445,CL317.contig2,Unigene9676）of S.litura under 150 mg·kg^-1Zn treatment were up-regulated,which were related to P450,hydroxybutyrate dehydrogenase,trypsin and esterase,respectively.After exposed to 600 mg·kg^-1Zn,GST1 proteins（Unigene28281and CL637.contig2）were up-regulated,while the genes or proteins related to alkaline phosphatase（Unigene818）and storage protein（CL2797.Contig1 and CL1523.Contig2）were down-regulated.4.After exposed to 150 mg·kg^-1Zn,the tolerance of S.litura to high temperature of32.5℃and 35℃improved,where the growth period shortened,the digestion and absorption ability and the growth and reproduction ability significantly enhanced.The possible molecular mechanism was that the expression of starch enzyme and protease related genes or proteins（CL1483.contig1 and Unigene28281）were up-regulated,which was helpful for nutrients digestion and absorption of larva to meet the energy needed for detoxification and growth.600 mg·kg^-1Zn stress and 35℃had antagonistic effects on the larvae,where the development duration of the larvae shortened,and the efficiency of conversion of ingested food（ECI）,efficiency of conversion of digested food（ECD）,RGR and the excretion rate of Zn increased.5.After exposed to 150 mg·kg^-1Zn,the tolerance of S.litura to CPM(7.00μg·m L^-1)enhanced,where the ECD,ECI,body weight and oviposition of S.litura larvae increased significantly.600 mg·kg^-1Zn stress and CPM had antagonistic effects,where life fitness of Zn treatment was similar with the control,while the ECI and ECD increased.After exposed to 150 mg·kg^-1Zn,the tolerance of S.litura larvae to CPF(10.59 mg·m L^-1),where the survival rate of larvae increased,the longvity of adults prolonged,and AD increased.600mg·kg^-1Zn stress and CPF had antagonistic effects,where no difference was found in the fitness between the two groups.The possible molecular mechanism of 150 mg·kg^-1Zn stress improving larvae tolerance to CPM and CPF was that the expression of CYP321B1（CL360.Contig1）related to P450s was up-regulated,which indicated that the detoxification ability of larvae increased.After exposed to 600 mg·kg^-1Zn,GST1,GSTO2,GSTe2,GSTs1 and GSTs3 genes or proteins that involves in glutathione S-transferase were down-regulated,and the expression of P450 related genes or proteins CL3369.contig2 and CL557.contig1（CYP6B50）was down regulated,which indicated that the detoxification ability of larvae decreased.In conclusion,the tolerance threshold of S.litura larvae to Zn stress was close to 450mg·kg^-1Zn.Under lower than 450 mg·kg^-1treatment,the absorption rate,excretion rate and food utilization efficiency of the larvae increased,which can effectively absorb Zn to meet the needs of insects;the tolerance to high temperature（32.5℃and 35℃）and pesticides（CPM and CPF）increased,which was related with up regulation of genes（such as CCE016a）and proteins（such as CYP321b1）involving in metabolism and detoxification.Under higher than 450 mg·kg^-1treatments,the growth and development of S.litura and its food utilization were significantly inhibited,because the protein expression involving in detoxification and growth was down-regulated.However,the antagonistic mechanism of600 mg·kg^-1Zn stress and high temperature or pesticide needs further study.