Molecular Mechanisms Of Summer Diapause Regulation In The Adult Galeruca Daurica

Galeruca daurica is a new pest with great outbreaks in the Inner Mongolia grasslands in recent years,and mainly feeds on Allium plants,such as Allium mongolium,A.polyrhizum,and A.ramosum.This pest has not only caused serious economic losses to grassland animal husbandry but also caused grassland degradation,which seriously threatened the ecological security in north China.Diapause is an adaptive strategy of insects to overcome adverse environmental seasons and ensures population continuity.Up to now,the physiological,biochemical and ecological mechanisms of insect diapause regulation have been deeply understood,but the molecular mechanisms of diapause regulation,especially the molecular mechanisms of obligatory summer diapause regulation,have been poorly understood.G.daurica,an obligatory diapause insect,occurs one generation each year,and oversummers as diapausing adults and overwinters as diapausing eggs.In this study,the molecular mechanism underlying summer diapause in G.daurica was intensively studied by proteomics,transcriptome and RNAi technology.This study not only helps to reveal the molecular regulation mechanism of obligatory summer diapause and reproductive diapause,but also provides a theoretical basis for developing pest control technology based on diapause regulation.The main results are shown as below:1.Using the isobaric tags for mass spectrometry-based proteome quantification（i TRAQ）technology to construct the proteomics database of the pre-diapause（PD）,the diapause（D）and post-diapause（TD）of G.daurica adults,and a total of 2383 proteins were obtained,of which 139 and 118 proteins were differentially expressed in the D/PD and TD/D comparisons,respectively.Bioinformatics analysis showed that the differentially expressed proteins were mainly related to phagosome pathway,metabolic process,stress response,cytoskeletal reorganization,insect juvenile hormone,calcium signaling pathway,ribosome pathway,and chemoreception.2.RNA-Seq was used to construct the transcriptome database for the adult G.daurica after the application of exogenous JH analogue（JHA）,methoprene.310 and 598differentially expressed genes（DEGs）were detected at 24 h and 48 h after JHA treatment,respectively.The KEGG analysis showed that a great number of DEGs were mainly enriched in various metabolic pathways,especially significantly enriched in energy metabolism,biosynthesis,stress response and signal transduction pathways.RNA-Seq and qPCR analysis indicated that JHA promoted the expression of Gd Vg,Gd FOXO and Gd Kr-h1 whereas suppressed the expression of Gd JHBP,Gd JHE,Gd JHAMT,Gd JHEH and Gd FAS,and its action intensity increased with the increase of JHA concentration.Injecting JHA before diapause can inhibit lipid accumulation and delay adults entering diapause,while applying JHA during diapause has no significant effect on diapause and lipid accumulation.3.The complete sequences of open reading frames（ORFs）of juvenile hormone esterase,methoprene-tolerant,juvenile hormone acid methyltransferase,juvenile hormone epoxide hydrolase,forkhead box O,vitellogenin,Krüppel homolog 1,and fatty acid synthase were cloned by RT-PCR,and named Gd JHE,GdMet,Gd JHAMT,Gd JHEH,Gd FOXO,Gd Vg,Gd Kr-h1,and Gd FAS,respectively.The ORFs were 222～6162 bp in length,encoding 74～2053 amino acids.qPCR analysis showed that GdMet,Gd JHE,Gd JHAMT,and Gd Kr-h1 were highly expressed at the pre-diapause stage,and the expression levels were significantly down-regulated during diapause,and the low-level expression pattern continued to the post-diapause stage.The expression levels of Gd Vg at the pre-diapause and post-diapause stages were significantly higher than that in diapause.Both Gd JHEH and Gd FAS were down-regulated at the pre-diapause stage,and both showed a trend of up-regulation first and then down-regulation during diapause,and the expression of Gd JHEH was up-regulated after diapause terminated,whereas Gd FAS was down-regulated.The expression level of Gd FOXO was highest on the 25^th day after eclosion,and remained at a low level in the other developmental stages.4.GdMet was silenced by RNAi,and the silencing efficiency was the highest（84.11%）at 48 h post-ds Met injection.The qPCR analysis showed that silencing GdMet inhibited the expression of Gd JHBP,Gd JHE,Gd Kr-h1 and Gd Vg,and promoted the Gd FAS expression but the expression of Gd JHAMT,Gd JHEH and Gd FOXO was not significantly affected.Compared with the control group（ds GFP）,the total lipid content in the ds Met-injected adults was significantly increased,which promoted fat body development,and finally caused the adults to enter diapause 3.1 d ahead.5.The full-length cDNA sequences of hexokinase and ribosomal protein S3a were cloned by RACE,and named Gd HK and Gd RpS3a,respectively.The results showed that the full-length cDNA sequence of Gd HK was 1699 bp,which contained a 1479 bp ORF encoding 492 amino acids.Gd HK was expressed at all developmental stages of G.daleruca,and the expression kept at the lowest level during adult diapause but rose abruptly to the highest level after diapause terminated.The expression level of Gd HK began to increase initially,but then decreased with the temperature increasing from 0℃to40℃.The full-length cDNA sequence of RpS3a was 800 bp with an ORF of 687 bp,encoding a protein of 228 amino acids.Gd RpS3a was expressed at different development stages with the highest expression levels after termination of adult diapause,and the lowest during the egg stage and adult diapause.Temperature had significant effects on the expression levels of Gd RpS3a with the highest level at 30℃and the lowest at 0℃.