Effects Of Imidacloprid Treatment On Gene Expression Related To Detoxification And Defense In Locusta Migratoria

Locusta migratoria manilensis(Meyen)is a model insect used in many reseraches.Imidacloprid,a neonicotinoid insecticide,has the benefits of the broad insecticidal spectrum,low toxicity and high efficiency.Serratia marcescens,a normal flora in the water and the soil,has high toxicity against the grasshopper Oedaleus infernalis Saussure with decently biocontrol effects.Thus,to investigate the effects of imidacloprid at different doses on gene expression related to detoxification,immune and digestive in L.migratoria midgut,the following experiments were performed in this study.1.Influence of high and low doses of imidacloprid on gene expression through transcriptome data analysisTo evaluate the effects of imidacloprid on gene expression in L.migratoria midgut,LD10(0.037 mg)and LDso(4.11 mg)as the low dose(LD)and high dose(HD)were selected to treat L.migratoria adults(half male and half female).In 24 h,the midgut was dissected from the survived insects and RNA was then extracted from the midgut sample.Illumina Hiseq2000 was used for the transcriptome sequencing.In three midgut transcriptomes,59331 unigenes were obtained,with the average length of 747 nt,and N50 reached 1187 nt.Unigene function annotates to NR,NT,Swiss-Prot,KEGG,COG and GO library with the number of 23201,11141,18837,16709,9818 and 11585,and all the annotated number of unigenes is 25531.Further analysis showed that the detoxification enzyme unigenes were more effected by imidacloprid low dose than that of high dose.Most differentially expressed unigenes of detoxifying enzymes distributed in CarEs A clade,GSTs sigma class and P450s 6 family.Most differentially expressed UGTs unigenes were down-regulated by imidacloprid.In unigenes related to immune,most lysozyme,diptericin,transferring and apoLp-? unigenes were down-regulated by both HD and LD treatments,while defensin-1 unigene was up-regulated by HD treatment.In the differentially expressed unigenes related to digestive enzymes,most 11 unigenes encoding chymotrypsin unigenes were differentially expressed.Among 11 chymotrypsin unigenes,8 unigenes were significantly down-regulated and 1 unigene was up-regulated.2.Validation of differentially expressed genes in midgut transcriptomes by quantitative real-time PCRBased on the differential expression ratios and unigene length,23 unigenes(corresponding to 23 genes)were selected to validate their differential expression under imidacloprid treatments.The results of qRT-PCR showed that 76.08%of validation results were consistent with that from the transcriptome.In P450s genes,CYP6K1(CL4043C2)was induced to 10.07 times by HD treatment,and to 9.32 times by LD treatment.CYP6BQ37(U27188)was induced to 6.68 times by HD,and LD had no significant effects on its expression.In constrast,,CYP6HQ1(CL659C2)was induced to 6.54 times by LD,while HD had no significant effects.In GSTs genes,GSTS2(CL1173C2)was induced to 9.00 times by HD,while LD had no significant differences.GSTD5(U33519),GST03(CL3618C4)and GSTS1(CL4610C5)were induced to 10.34,8.34 and 3.04 times respectively by LD,while HD had no significant differences.In CarEs genes,CesA8(U12483)was induced to 3.37 and 5.46 times by HD and LD respectively.CesD1(U30920)and CesA20(U6549)were induced to 7.03and 13.32 times by HD,while LD had no significant differences.In constast,CesA3(CL3747C1)was induced to 4.50 times by LD,while HD had no significant differences.The results indicated that the differential expressions of some detoxification enzyme genes were dependent on the doses of imidacloprid used.Nine genes involved in immunity system were also selected,and 88.89%of them were consistent with the results from the transcriptome.Mucin-5(CL2786C1)was induced to 12.2 times by HD,and to 9.23 times by LD,with the highest differential expression ratios.PGRP1(CL2306C2)was induced to 11.67 times by HD,and to 7.96 times by LD.Serpin4(CL2600C2)was induced to 7.18 times by HD,and to 10.08 times by LD.Defensin-1(U43570)was induced to 5.17 times by HD,while HD had no significant differences.Diptericin(U8926)was down-regulated by both HD and LD,with the ratio of 0.25 and 0.12 compared to the control.3.The effects of low dose of imidacloprid and Serratia marcescens treatment on hemolymph immune pathway genes of L.migratoriaTo better understand the influence of imidacloprid on the expression of L.migratoria immunity related genes,some genes were selected to examine their expression in hemolymph following imidacloprid(low dose,LD)treatment and compared to that of the treatment by Serratia marcescens,a strain isolated previously by our laboratory with high pathogenicities to a range of insect speices.Three genes,PGRP-LE,Relish and Diptericin,related to IMD and other three genes,GNBP3,MyD88 and Defensin-1,related to Toll pathways were examined.PGRP-LE and Relish were up-regulated to 6.06 times and 9.50 times by S.marcescens,wihle Diptericin showed no significant difference.Interestingly,GNBP3was also overexpressed to 4.94 times following S.marcescens treatment.LD imidacloprid treatment increased the expression of Relish and Diptericin to 6.77 and 7.88 times of the untreatment control.In both S.marcescens and imidacloprid treatments,Defensin-1 was significantly down-regulated.The results showed that imidacloprid could regulate gene expressions in L.migratoria IMD pathway,but show no obvious influence on gene expressions in Toll pathway,which suggests that the integrated effects on insect immune system by both chemical insecticides and insecticidal microorganisms should be in consideration when two kinds of insecticides are mixed to control insect pests.