Study On The Mechanism Of Tolerance To High Concentration Of Ecdysteroid In Spodoptera Litura

The ecdysteroids is essential to the development of insects.Metamorphosing insects regulate the molting and metamorphosis of insects by precisely controlling the ecdysteroids titers.Insects can not synthesize ecdysteroids from scratch,but plants can.Some insects feed on plants that contain ecdysteroids and cause large amounts of ecdysteroids enter into the body.Different insects have different levels of ecdysteroids sensitivity.Insects such as silkworm and other insects are very sensitive to ecdysteroids,while the omnivorous agricultural and forest pest Spodoptera litura can tolerate high concentrations of exogenous ecdysteroids.However,the mechanism of tolerance in S.litura to high concentrations of ecdysteroids is unclear.In this study,the effects of 20-hydroxyecdysone(20E,highly active ecdysteroids)on S.litura larvae were investigated.The results of the study are as follows:1.Effect of ingestion and injection of 20 E on growth and development of S.litura.The effects of 20 E on S.litura larvae were investigated by feeding 10 ?g,20 ?g,50 ?g 20 E,and injected 10 ?g,20 ?g 20 E for each larvae,respectively.20% ethanol were used as a control.The body weight,pupa time,and pupa rate of S.litura larvae before and after treatment were measured.The results showed that from the larvae to the adult,the growth and development status of the control group and the treatment group were the same.There were no significant differences in body weight,pupation time and pupation rate between the treated larvae and the control group,indicating that high concentrations of 20 E had no significant effect on the growth and development of S.litura.2.Detection of 20 E related substances in different tissues and excretions of S.litura after treatment.High-performance liquid chromatography was performed on the midgut,the contents of the midgut,the blood at 1 h and 3 h after treatment.The same survey was performed for the feces after 8 h,24 h,and 48 h of the treatment.It was found that feeding 20 ?g of 20 E into the midgut after 1 h of the treatment was metabolized on the inner surface of the midgut.After 3 hours of the treatment,the basic metabolism is completed and released into the intestinal lumen for further transformation.Feeding 20 E was completely metabolized to final product after 8h of treatment and excreted with feces.3.Analysis of differentially expressed genes in midgut transcriptome after the treatment.In this study,RNA-seq was used to perform transcriptome sequencing of the midgut after 3h of feeding 20?g 20 E and 20% ethanol.After quality control analysis,a total of 31.6 Gb of effective bases in the transcriptome were obtained.The genome of S.litura was assembled and a total of 13,946 genes were assembled,of which 11851 genes could be translated into proteins.Through screening,865 differential genes in the midgut were obtained,of which 405 were down-regulated and 461 were up-regulated.GO(Gene oncology)enrichment got two types of steroid hormone-related gene upregulation.These differentially upregulated genes are those involved in the metabolism and response of ecdysteroids.This indicates that the ecdysone response genes were up-regulated after feeding 20 E.It also caused the up-regulated expression of ecdysteroids metabolism-related genes,resulting in metabolically feeding high concentrations of 20 E.I further performed binding site predictions on the promoters of the three great different ecdysteroids metabolism genes.I found that the most possible transcription factor is Ec R::USP(The complex of Ecdysteroids receptor and Ultraspiracle).There are also binding sites for transcription factors such as Br C-Z2(Broad complex zinc-finger 2)and FTZ(Beta Fushi Tarazu-Factor 1).These transcription factors and genes are up-regulated.It can be speculated that these transcription factors regulate the up-regulated expression of these three genes and thus metabolize high concentrations of 20 E.In summary,this study focused on tolerance of S.litura larvae to ecdysteroids.The results indicated that there was no significant effect on body weight and pupation time of S.litura by ingesting and injecting with high concentrations of 20 E.After feeding 20 E for 1 h,it entered the intestine cavity,causing ecdysteroids response and metabolic gene up-regulation.After 8 h,20 E is metabolized into the final product in the midgut and excreted.It is this rapid and effective metabolic mechanism that leads to the tolerance of S.litura to exogenous high concentrations of 20E.