| Diapause is a physiological process of growth and metabolic arrests that is often influenced by adverse environmental conditions in insects.Insect diapause is a complex and dynamic process,including the diapause induction,preparation,initiation,maintenance,termination,and post-diapause quiescence.Diapause preparation is crucial for insect diapause maintenance and termination,post-diapause development.Diapause-destined(DD)individuals transfer environmental cues from diapause induction to preparation stages,and the decision of whether to enter diapause is made.Furthermore,DD individuals ‘decide' to increase their energy accumulation during diapause preparation stage.However,the molecular mechanisms of major events during diapause preparation poorly understand.The cabbage beetle Colaphellus bowringi Baly is a serious pest of cruciferous vegetables and undergoes diapause as adults in soil in both summer and winter.Temperature or photoperiod cues are the main factors of diapause induction.The larva stage of C.bowringi is sensitive to diapause induction signaling.Diapause preparation stage is very short and diapausing beetles exhibit digging behavior,the cabbage beetle is an excellent species in which to investigate the mechanisms regulating diapause preparation.The Xiushui population(29°1?N,114°4?E)was used in this study.We firstly established a C.bowringi transcriptome and screened valid reference genes,based on this valuable database platform,isobaric tags for relative and absolute(iTRAQ)and RNA-Seq technologies were used to identify differential expression proteins and genes during diapause preparation stage,RNA interference(RNAi)and microinjection exogenous juvenile hormone(JH)methods were used to analyze the molecular mechanism of major events during diapause preparation stage.The main conclusions are as follows:1.A de novo transcriptome and valid reference genes for quantitative Real-Time PCR in C.bowringiBecause of the lack of genomic information and valid reference genes,the knowledge on the molecular bases of the physiological processes is limited in C.bowringi.In this study,using Illumina sequencing platform,more than 57 million sequence reads derived from C.bowringi were obtained,which were assembled into 39,390 unique sequences,and average length is 792 bp.A total of 24,070(61.11%)Unigenes can be annotated with biological functions.9,048 of these sequences were obtained a Clusters of Orthologous Groups(COG)classification,covering 25 categories,and 16,951 wereassigned to 255 Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways.Base on the transcriptome,the sequences of 11 candidate reference genes(GAPDH,RPL32 e,RPL19,EF1?,TBP,TBP1,ACT1,ACT2,?TUB,?TUB1,?TUBC)were identified by reverse transcriptase polymerase chain reaction.RPL19,ACT1,and EF1? proved to be the most stable reference genes.RPL19 and ACT1 can be used in the analyses of molecular mechanisms regulating diapause preparation in C.bowringi.The great number of gene sequences and valid reference genes provide a valuable platform for investigating the molecular mechanisms during diapause preparation process in this beetle.2.Using iTRAQ to detail the diapause-preparatory proteome of the beetle C.bowringi and identify fatty acid-binding protein functionIn the cabbage beetle,photoperiodic cues perceived during the larval stage are stored in the head and later used to prepare adults for diapause.Lipid metabolism signalling may be a potential pathway for the transmission of environmental cues from diapause induction to preparation stages.Therefore,i TRAQ technology was used to quantitatively compare the expression profiles of proteins identified during the onset of diapause preparation stage in the heads of adult female C.bowringi.What's more,the function of fatty acid-binding protein(FABP)was analyzed.A total of 3,175 proteins were identified,297 of which were differentially expressed between DD and non-diapause-destined(NDD)female adults,and 141 proteins were upregulated in DD female adults,156 proteins were downregulated.After COG classification,many abundant of these upregulated differential expression proteins(DEPs)were involved in energy production and conversion,carbohydrate metabolism and transport,and lipid metabolism.FABP was assigned to both the ‘peroxisome proliferator-activated receptor' signaling pathway and the ‘fat digestion and absorption' pathway.After silencing FABP during the onset of diapause preparation stage,the hypertrophy of fat body was inhibited and the fat body became detached,the number of lipid droplets was significantly decreased.Moreover,the decreased lipid storage regulated Heat shock proteins transcript abundance.The results suggest that FABP playing vital roles in lipid metabolism could regulate stress tolerance genes expression,which are involved in regulating diapause preparation.The high number of DEPs identified could provide novel insights into the molecular regulation of diapause preparation in insects.3.The regulation of nutrition metabolism genes by juvenile hormone signaling contributes to reproductive diapause preparation in C.bowringiWhether JH signaling regulates adult diapause via regulating nutrients metabolism poorly understand.This study used RNA-Seq technology to quantitatively compare the differential expression genes(DEGs)between diapause preparation and pre-oviposition stages.The results showed that after female adults feeding for 0 to 2 days,DEGs in DD and NDD individuals were both range from 800 to 1300.After female adults feeding for 2to 4 days,173 DEGs were upregulated and 152 DEGs were downregulated in NDD individuals,however,2,576 DGEs were upregulated and 1,998 DGEs were downregulated in DD individuals,indicating that the physiological process of diapause preparation is more complicated.KEGG pathway analysis showed that DEGs were mainly assigned to carbohydrates,lipids and proteins metabolism pathways,showing that nutrition metabolism regulation is the main characteristic during diapause preparation and pre-oviposition stages.Glycolysis,pentose phosphate,fatty acid biosynthesis,and glycerolipid metabolism pathways were more active during diapause preparation stage,whereas,ribosome pathway was more active during pre-oviposition stage.These results are well consistent with the nutrition metabolism characteristic between reproductive and diapause individuals.JH signaling was active during pre-oviposition stage,whereas,in the resting state during diapause preparation stage.The results showed that JH acts through Met to facilitate the reproductive and diapause by regulating nutrition metabolism genes.JH-Met signaling suppresses the carbohydrates and lipids biosynthesis in DD individuals,and promotes protein synthesis in DD individuals.4.Fatty acid synthase 2 contributes to diapause preparation in adult female C.bowringi by regulating lipid accumulation and stress tolerance genes expressionIt has been demonstrated that fatty acid synthase(FAS)plays a central role in lipid accumulation during diapause.However,whether other important aspects of diapause,such as stress tolerance,the duration of diapause preparation,and the incidence of diapause itself,are regulated by FAS remains unknown.A search of C.bowringi transcriptome found two FAS genes,FAS1 and FAS2.On the Simple Modular Architecture Research Tool and NCBI Conserved Domain Search websites,the protein sequence of FAS1 had seven functional domains,whereas,the protein sequences of FAS2 contained nine functional domains.A rooted phylogenic tree placed FAS1 and FAS2 on two distinct,major branches.In DD female adult,the mRNA expression pattern of FAS2 varied muchmore obvious,and FAS2 was more highly expressed than FAS1 after feeding for 2 days.What's more,the mRNA expression level of FAS2 of DD female adult was significant higher than that of FAS2 of NDD female adult at the same time.These suggest that FAS2 may be more important than FAS1 during the diapause preparation stage.FAS2 knockdown suppressed lipid accumulation,the hypertrophy of the fat body was inhibited and the fat body became detached,triacylglyceride content was significantly decreased.The decreased lipid accumulation affected resistance of oxidative stress and water content.However,silencing FAS2 had no significant effects on the duration of diapause preparation or the incidence of diapause.FAS2 transcription was suppressed by JH and JH receptor Met.These results suggest that the absence of JH-Met induces FAS2 expression,thereby promoting lipid accumulation and regulating stress tolerance genes expression and water content in DD female beetles.In present study,a substantial dataset of transcriptome was established and valid reference genes were screened,these established a valuable platform for gene expression analysis in C.bowringi.The large number of DEPs and DEGs identified during diapause preparation stage could enrich the molecular resources of this beetle,and contributes to the study of molecular mechanisms of diapause regulation in C.bowringi.The results showed that JH-Met signaling contributes to reproductive and diapause through regulating nutrition metabolism gene transcription.In addition,the variation of lipid accumulation could affect stress tolerance genes expression and water content during diapause preparation stage.These studies provide novel insights into the molecular regulation of diapause in insects. |
PDF Full Text Request