Biochemical And Molecular Mechanisms Of The Toxicological Effects Of Methoxyfenozide To Lymantria Dispar

The common lymantriidae.Gypsy moth(Lymantria dispar), was a worldwide distributed pest. Frequent application of chemical insecticides against Lymantria dispar had led to the development of insecticide resistance. Methoxyfenozide which was a novel insecticide acting in a similar fashion of ecdysone was found to produce phenotypic effects that finally interfer the formation of new larval cuticle in insects. Methoxyfenozide showed excellent insecticidal activity against larval of Lepidoptera and high degree of safety to nontarget organisms,and is now used as an ideal candidate to substitute organophosphate and pyrethroid insecticides.Mixed-function oxidases (MFOs),as the detoxifying enzymes in insects,could participate in all kinds of insecticide detoxification.Its substrate was widespread and was one of the important ways to metabolize pesticide. Amidase was thought to participate in pesticide hydrolysis of aromatic amide groups.It probably played an important role in the insecticide resistance. The ecdysone receptor (EcR) and ultraspiracle (USP) were the nuclear receptors and ecdysone molecular targets found in insects. Based on the gypsy moth as the object, we determinated the activity of key enzyme targets MFOs and amidase of larvaes were treated with methoxyfenozide. On this basis,we also explored the effects of methoxyfenozide on target genes EcR and USP, which will clear toxicology mechanism of biochemistry and molecular.lt has important significance and the findings were as follows:1、To explore the insecticidal activity and the effects of methoxyfenozide on the enzyme activities related to the metabolism in insects,cabbage leaf dip method was used to finish the toxicity test and determine the activities of MFOs and amidase in2nd and3rd instar larvae of Lymantria dispar which were treated with methoxyfenozide. The results showed that48h-LCso values of methoxyfenozide were14.004for the2nd instar larvae and24.105mol/L for the3rd instar larvae,48h-LC5values of1.573and2.090mol/L respectively,48h-LC20values of4.575and6.898mol/L respectively. After the larvae were treated with two sublethal concentrations48h-LC5and48h-LC20of methoxyfenozide,the activity of MFOs in the2nd instar larvae increased first and then decreased and increased again with the prolonged time. However,it had the opposite effect on the3rd instar larvae after treatment. The activity of amidase was significantly induced and inhibited,taking turns to appear alternately by the methoxyfenozide treatment. It is concluded that the methoxyfenozide has a high level of toxicity against the2nd and3rd instar larvae,interference effect on MFOs and amidase signally by methoxyfenozide.2、Through the analysis of the gypsy moth transcriptome library,we got4genes of LdEcR and1gene of LdUSP. The gene LdUSP was full-length gene, its reading frame (ORF) length of 1395bp and encoded464amino acids, and it didn’t contain the signal peptide sequence;Prediction according to the conserved regions of the gene LdUSP protein sequences belong to the insect nuclear receptor superfamily members;These genes by multiple sequence alignment and phylogenetic tree analysis,LdEcR-5567and LdEcR-11596had a higher homology between citrus swallowtail butterfly (Papilio xuthus)(BAM20285.1) and Indian meal moth(Plodia interpunctella)(AAR84611.1) than red monarch butterfly (Danaus plexippus)(EHJ66465.1);The gene LdEcR-21633and LdEcR-4289and beet armyworm (Spodoptera exigua)(AFK27931.1) had recent genetic relationship.3^Using the Real-time RT-PCR,we analyzed the specific expression of ECR and USP genes in each developmental stage and stress response of methoxyfenozide sublethal concentration (LC5and LC20).The results showed that as opposed to the1st instar larvae, addition to gene LdUSP-23169decreased expression in the4th instar, pupae and male developmental stages and the expression of LdEcR-5567was also reduced in the3rd instar and5th instar developmental period;LdECr-27633showed high expression in all developmental stages. In the egg stage, relative to the1st instar larvae, the gene LdUSP’-23169and LdEcR-27633,4289,5567.11596,the expression levels of performance for highly expressed. In the larval stage, the five genes in the adult stage higher expression.The mRNA transcriptional levels of genes LdEcR and LdUSP changed remarkably after sublethal concentrations of methoxyfenozide in2nd and3rd larvae. By two concentrations treated, the mRNA transcription level of gene LdEcR-23169was significantly higher than the expression of other genes in2nd instar larvae,the expression of gene LdEcR11596was always suppressed by LC5dose treated.Howere,the expression of gene LdEcR and LdUSP significantly inhibited in the3rd larvae after two concentration treatment, LdEcR-4289gene expression was also always suppressed.From the above results can be seen MFOs and amidase were important biochemical taret enzyme.The gene LdUSP-23169and LdEcR-27633played a major role in response to methoxyfenozide stress for2nd and3rd larvae. These results will provide a theoretical basis to further explore how to regulate and control the molecular mechanism of EcR and USP.