Hormone-Related Pathway And Its Regulation On Gene Expression During Metamorphosis In The Silkworm, Bombyx Mori

Insects are the majority of animal species in the world.Most insects must undergo the molting and metamorphosis in order to complete their entire life cycle.In general,the shape,structure and living habits of these metabolous insects change dramatically during molting and metamorphosis and form four different developmental stages including egg,larva,pupa and adult.Molting and metamorphosis are controlled by molting hormone(MH)and juvenile hormone(JH)and regulated by a large amount of genes.The studies of molecular mechanism of hormone regulation on metamorphosis are of great significance to elucidate the genetic basis of the growth,development and evolution of insects.Silkworm,Bombyx mori is a member of the holometablous insects.It is an important insect economically,and is also one of the best-characterized model insects with solid research basis and numerous mutant lines and breed lines.The studies on the hormone regulation on silkworm metamorphosis can not only accelerate the elucidation of regulatory mechanism of silkworm metamorphosis and help to well understand of insect metamorphosis and genetic basis of biological evolution diversity,but also provide important clues for the artificial regulation of silkworm development and biological control of pests.With the advanced achievements in other insects,whole genome sequence and microarray of silkworm,we performed the identification of genes involved in the hormone-regulated pathway of silkworm metamorphosis,and surveyed genome-wide gene expression during metamorphosis,and hence,explored the relationship between gene expression and the hormone regulation during metamorphosis.The main results present as follow:1.Juvenile hormone-related genes in silkworm genome We have used the sequences of the known genes involved in JH biosynthesis,metabolism and signal transduction as queries to search against the silkworm genome sequences.As a result,43 JH-related genes were identified in silkworm,of which,seven genes(Acat,HMGS,HMGR,MevK, MevPK,MevPPD,and IPPI)participate in upstream MVA pathway of JH biosynthesis and catalyze acetyl-CoA to generate isopentenyl diphosphate(IPP),FPPS1-2 catalyzes IPP to generate farnesyl pyrophosphate(FPP).Thirteen genes(SDR,FAMeT1-4,JHAMT1-7,and Cyp15a1)involve in downstream isoprene branch of JH biosynthesis.Another three genes(AT,AS,and AlstR)are associated with neuropeptide regulation on JH biosynthesis.Besides,twelve genes(JHE1-3, JHEH1-6,and JHDK1-3)and six genes(JHBP,Met,EcR,USP,FKBP39,and Chd64)are involved in the metabolism and signal transduction of JH,respectively.In addition to AlstR,other JH-related genes can be located on different chromosomes by using SNP markers.What's more,multiple copies of each of five genes(FPPS,JHAMT,JHE,JHDK,and JHEH)clustered together.And these, as a whole,would provide important and valuable information for future investigation on gene functions.Comparative analysis found that each of genes involved in the MVA pathway was single in B.mori,Drosophila melanogaster,Anopheles gambiae,Apis mellifera,or Tribolium castaneum, suggesting this upstream pathway of JH biosynthesis was conserved in these insects.FPPS was evolved into two types with different functions in Lepidoptera and is a key enzyme that dominates the types of JH among insects.The copies of each gene involved in isoprene branch of JH biosynthesis and in JH metabolism were greatly diverse,and this may be associated with various types of JH among insects.Besides,each of genes participated in JH signal transduction was also single,speculating that the pathway of JH action was highly conserved in different insects.We utilized the microarray data of gene expression in multiple tissues of silkworm on day 3 of fifth larval instar to survey the expression patterns of JH-related genes.And,the results showed that the majority of genes related to JH synthesis were low expressed.The expression of genes involved in JH metabolism was relatively higher and tissues-specific,which indicated that the process of JH biosynthesis and metabolism was inhibited and induced on day 3 of fifth larval instar,respectively.It suggested that these expression changes of JH-related genes leaded to the decline of JH titers.2.Nuclear receptor gene family in silkwormNuclear receptors(NRs)are one of the most abundant classes of transcriptional factors in animals. Many members of this superfamily are the receptors of MH or are induced by MH in insects and are directly involved in the MH regulation of insect molt and metamorphosis.Based on the homology search,19 NR genes were identified in the silkworm genome,14 of which contain a typical DNA-binding domain(DBD)and ligand-binding domain(LBD),and 15 members can be located on different chromosomes.In addition,the similarity between LBD sequences in different NRs was significantly lower than that of DBD.We also found that 18 members of silkworm NRs can be identified 1:1 orthoiogs in D. melanogaster,A.gambiae,A.mellifera,or T.castaneum.But,no syntenic blocks are found among these five insects.DBD- and LBD-based phylogenetic analysis showed that the ortholog of gene coding for each of NRs from all insects always firstly clustered together,suggesting that insect NRs originated before radiation of five insect species.On the other hand,among the 15 NRs containing LBD,the average number of introns in per NR gene in B.mori is 3.2,which is higher than that in other four insects.This diversity in the key functional domain speculated that potential changes of the function of NRs may occur during insect evolution.We used RT-PCR and microarray data to survey the spatial and temporal expression patterns of the silkworm NR genes.The results showed that several NR genes were specifically expressed at different developmental time points during metamorphosis.BmβFTZ-F1 was up-regulated before spinning and pupation,which is in accordance with the pulse of MH during silkworm metamorphosis.Three genes,BmEcR-B,BmUSP-2,and BmHR3,were subsequently expressed after the pulse of MH and displayed a MH-induced cascade.Moreover,according to the microarray data of gene expression in multiple tissues of silkworm on day 3 of fifth larval instar,we found that BmE75,BmHR38,and BmHR78 were expressed in the gonads,head,and integument at this stage; BmEcR was expressed in the midgut,integument,and fat body;and BmDSF was highly expressed in the midgut,integument,fat body,and ovary.The relationship between the expression patterns and functions await investigation.3.Microarray analysis of gene expression during silkworm metamorphosisIn order to explore hormone regulation on gene expression during silkworm metamorphosis,we have used silkworm whole genome microarray with 22987 probes to investigate gene expression profiles at 19 developmental time points during metamorphosis.As a result,a total of 12097 genes were detected at one time points at least,and eventually,7628 genes were expressed differentially in all selected time points.We first analyzed the expression changes of the hormone-related genes during metamorphosis. The results showed that 31 of 43 JH-related genes were detected to be expressed.And,most of genes coding enzymes involved in MVA pathway were expressed during pupa-adult transition,and the two types of BmFPPS were not only expressed at the later of pupa development and adult stage,but also expressed only in the females.Majority of the genes that involved in the downstream pathway of JH biosynthesis were also expressed at the later of the pupa.Hence,we speculate that JH would be biosynthesized at the later of female pupa and the adult period,and this may be related to the regulation of oogenesis during this period.In addition,11 of 19 NR genes were also detected. BmEcR and BmUSP genes were expressed on the day 7 of 5th instar and may be related to the regulation of wandering.BmHR3 and BmβFTZ-F1 were highly expressed at 36 and 48 hours(just completing spinning)after wandering,respectively.This indicated that BmβFTZ-F1 may be associated with onset of silkworm metamorphosis and hence would be transcriptionally regulated by BmHR3.Also,among genes coding enzymes involved in MH biosynthesis,BmCYP302A1 and BmCYP315A1 were upregulated from 48 hours after wandering to adult,and BmCYP314A1 was upregulated at 120 hours of wandering(day3 after pupation),which is coincide with the pulse of MH during pupa-adult transition.Comparative analysis found that 577 genes were highly expressed in the male,in which a-tubulin,β-tubulin,testes-specific protein tektin,and malate dehydrogenase were correlated with testis development or spermiogenesis.352 genes were highly expressed in female and most of them were only expressed in the latter of pupa development,which may be closely related to the oogenesis. Importantly,617 genes were highly expressed before wandering,and 476 and 99 genes were highly detected after 48 hours of wandering(just completing spinning)and during spingning/prepupa, respectively.Among the genes that are highly expressed prior to wandering,JH esterase and JH epoxide hydrolase are associated with the degradation of JH.Arylphorin,arylphorinβsubunit,low molecular weight 30 kDa protein,and 30 kDa protein 19G1 and 21G1 are related to the storage of the nutrients.Most of the genes expressed after the completion of spinning have enzyme or structural molecular activity,and may participate in histolysis and histogenesis.Interestingly,in the region of 2000bp upstream of 8 cuticle proteins genes expressed at this stage,we identified cis-regulatory motifs for BmβFTZ-F1 that also was highly expressed at this same stage.Among gene expressed during spinning/prepupa,yellow protein may be involved in chromogenesis.And,serpin K, serpin-SB,and antitrypsin belong to serine protease inhibitors and they may play key roles in protecting fibroin from being degradated by various proteases.4.The influence ofβFTZ-F1 and Met genes on silkworm metamorphosisβFTZ-F1 and Met are MH receptor and potential JH receptor.We performed RNAi of BmβFTZ-F1 during silkworm metamorphosis and obtained developmental defective phenotypes in the RNAi treated group.RT-PCR analysis revealed that several cuticle protein genes were down-regulated in defective phenotypes,strongly suggesting BmβFTZ-F1 was critical for silkworm metamorphism and regulate the expression of cuticle protein genes.Also,we cloned full-length cDNA of JH potential receptor.Multiple sequence alignment showed that Met was conserved among different insect species.RT-PCR experiment confirmed that BmMet was expressed in the entire life cycle and different tissues on day 3 of fifth larval instar.The ubiquitous expression patterns indicted that BmMet may be a cofactor in JH signal transduction pathway,or a member of JH receptor complex.5.Hormone regulation of silkworm metamorphosisIn summary,various types of JH in silkworm were correlated with multiple copies of JH-related genes especially FPPS.The high expression of JH metabolism-related genes in the late fifth larva declines titer of JH at this stage.Genes involved in JH biosynthesis were re-expressed in the late female pupa and adult,suggesting that JH may be also biosynthesized at this stage and may be related to the formation of eggs.BmMet as potential JH receptor was expressed throughout silkworm development and may be only a cofactor of JH action.What's more,the genes coding enzymes control MH biosynthesis were expressed before the completion of spinning,then continued to later pupa stage,and finally rise the titer of MH in pupa.The expression pattern of BmEcR-B,BmUSP-2, BmHR3 and BmβFTZ-F1 displayed MH-induced cascade.We also discovered the recognition motif for BmβFTZ-F1 in the UTR of cuticle protein genes and observed the up-regulated expression before pupation and down-regulated expression after RNAi of BmβFTZ-F1,suggesting that the cuticle protein genes associated with pupation was regulated by BmβFTZ-F1.However,the relationship between the stage-specific expression of more genes during silkworm metamorphosis and the regulation of JH or MH awaits further elucidation.