The Regulation Of Chico-drk Signaling Branch To Larval Metamorphosis In Leptinotarsa Decemlineata

Leptinotarsa decemlineata(Say)is a notorious quarantine insect pest.In many potato producing areas around the world,defoliation by the beetle can cause severe yield losses in potato crops.It is well known that insulin/insulin-like signaling and target of rapamycin(IIS/TOR)signaling controls body size,by regulating growth rate and growth duration in both holometabolans and hemimetabolans.Moreover,IIS/TOR is also critical for survival and metamorphosis of holometabolan insect larvae.However,the molecular mechanism remains undetermined.In insects,ILPs binding to insulin receptor(InR),the activated insulin receptor(InR)and insulin receptor substrate(Chico)complex recruits downstream of receptor kinase(Drk)and phosphatidylinositol-3-kinase(PI3K)to initiate two separate signaling branches,i.e.,Drk-mitogen-activated protein kinase(MAPK)and Pi3K-protein kinase B(PI3K-PKB)cascades.The aim of this thesis is to explore the key factors regulating metamorphosis in IIS/TOR signal pathway.A key IIS/TOR gene Ldchico,a PI3K-PKB branch gene Ldpi3k,a DRK-MAPK gene Lddrk,and a food consumption related gene LdsNPF were identified.We used RNA interference(RNAi)to knock down each gene and a combination of them.We found that the IIS/TOR signaling cascade branch,i.e.,Chico-Drk-MAPK pathway,is responsible for the control of larval metamorphosis.The main results were showed as follows.1.Identification and cloning of key genes in IIS/TOR signaling.We identified three important genes,Ldchico,Ldpi3k and Lddrk.The full-length sequences were obtained,and the phylogenetic trees were constructed based on amino acid sequences.The gene structures were drawn according to the genomic data.Ldchico has a 3498 bp open reading frame encoding 1166 amino acid residues.LdChico has a typical PH and PTB domains.Ldpi3k92E possesses a 3219 bp open reading frame which codes for a protein with 1072 amino acid residues.LdPI3K92E has a p85 binding site,a RAS binding site,a C2 domain,a PIK domain and a type I PI3K catalytic domain.Lddrk has a 636 bp open reading frame that encodes 211 amino acid residues.The LdDrk includes two SH3 domains and a SH2 domain.The identification facilitates our further researches on their functions.2.Knocking down Ldchico affects the metamorphosis of L.decemlineataIn this chapter,we further observed that the expression of the insulin receptor substrate gene chico(Ldchico)and the phosphoinositide-3-kinase gene pi3k(Ldpi3k92E)was repressed in LdILP2 depleted larvae.We examined the temporal and spatial expression profiles of Ldchico and Ldpi3k92E,and found that both were expressed throughout all developmental stages.The expression level of Ldchico peaked 36 h after ecdysis to the fourth-instar larvae and sharply decreased thereafter,while the mRNA level of Ldpi3k92E peaked 12 h after ecdysis and was then gradually reduced.Ldchico was highly expressed in the Malpighian tubules,hindgut,fat body,muscle and ventral ganglion,while Ldpi3k92E was abundantly transcribed in the brain-corpora cardiaca-corpora allata complex,Malpighian tubules and hemocytes.In order to identify key genes affecting metamorphosis in the IIS/TOR pathway,we knocked down Ldchico or Ldpi3k92E.RNAi of Ldchico or Ldpi3k92E upregulated the expression of InR and hindered the IIS/TOR pathway,decreased food consumption,and affected absorption and metabolism of amino acids and sugars.Compared with the control larvae,the concentration of the total amino acids in the RNAi larvae was similar.In contrast,the contents of arginine,asparagine,glutamate,glutamine,lysine and proline were higher,whereas the amounts of histidine,isoleucine,leucine,methionine,phenylalanine,serine,tyrosine and valine were lower in either Ldchico or Ldpi3k92E RNAi specimens.Consistently,we found the expression levels of genes encoding three serine proteases(intestains,IntB4/D4/E2),a cysteine(chymotrypsin-like,SPS1A),and a nutrient amino acid transporter(LdNAT1)were significantly reduced in either Ldchico or Ldpi3k92E RNAi larvae.Glucose and trehalose concentrations were significantly increased and glycogen quantities were lower in Ldchico or Ldpi3k92E depleted larvae,compared with those in control counterparts.In agreement with the findings,the expression levels of trehalose metabolism genes,LdTPS,LdTRE1a,LdTRE1b and LdTRE2 were reduced in Ldchico and Ldpi3k92E RNAi beetle larvae.The transcript levels of a glycogen synthase gene(LdGS)were higher in the RNAi samples than those in PBS-and dsegfp-fed larvae,whereas the mRNA levels of a glycogen degradation gene(glycogen phosphorylase,LdGP)were similar in Ldchico and Ldpi3k92E RNAi specimen to those in control counterparts.Moreover,RNAi of Ldchico or Ldpi3k92E reduced expression of several 20E(LdEcR,LdHR3 and LdE75)and JH(LdJHAMT,LdKr-hl and LdHairy)signaling genes.As a result,larval development was postponed and larval growth was inhibited.Intriguingly,knockdown of Ldchico,rather than Ldpi3k92E,impaired larval-pupal and pupal-adult ecdysis,and specifically repressed transcription of another 20E signaling gene LdUSP.For Ldchico depleted larvae,ingestion of JH rescued the expression level of LdKr-h1 and the decreased weight,dietary introduction of 20E rescued the expression of LdEcR,LdHR3 and LdE75,but not restored neither the decreased LdUSP mRNA level,nor the reduced pupation and adult emergence rates.Therefore,Chico is critical for the regulation of larval-pupal-adult transition by a PI3K-independent pathway,perhaps through activation of USP in L.decemlineata.3.Drk-MAPK affects potato beetle metamorphosisIn this chapter,we noted that silencing LdILP2,Ldchico or Ldpi3k92E did not decrease the expression level of Lddrk.We examined the time and spatial expression profiles of Lddrk and found that the Lddrk level was high in the eggs and larvae.The mRNA level was gradually decreased in wandering prepupae and pupae,and reached its trough at adult stage.Since Drk mediates the function of the receptor tyrosine kinase,we examined the temporal and spatial expression profiles of 8 receptor tyrosine kinases involved in insect development.Among the receptor tyrosine kinases involved in insect development,only two InRs and Torso were highly expressed in the final larval instars.Knocking down Lddrk did not reduce food consumption,not affect the maximum weight,absorption and metabolism of amino acids and sugars.In the Lddrk RNAi larvae,the expression levels of LdIntD4 and LdNAT1 showed no significantly difference from controls;the concentrations of glucose,trehalose and glycogen were similar with the control counterpart;the expression levels of the trehalose metabolism genes LdTPS,LdTRE1a,LdTRE2,LdGS,and LdGP showed no significantly difference with those in controls.RNAi of either Lddrk or Ldchico,or both of them equally impaired larval-pupal-adult transition,and similarly repressed the expression of representative MAPK(Ldras and Ldraf),ecdysteroidogenesis(Ldphm and Ldsad),and 20E signaling(LdEcR,LdUSP,LdHR3 and LdE75)genes.20E ingestion by Lddrk RNAi larvae completely restored the reduced mRNA levels of LdEcR,LdHR3 and LdE75,but rescued neither the decreased Lddrk and LdUSP levels nor the lowered pupation and emergence rates.Thus,our findings suggest that the Drk-MAPK cascade is involved in meta-morphosis regulation in L.decemlineata.4.Chico or pi3k affects food consumption through sNPFIn this chapter,we found that knocking down Ldchico or Ldpi3k92E did not affect the expression of Ldsk,but significantly decreased the expression of LdsNPF.In the dsLddrk larvae,the mRNA levels of Ldsk and showed no differences from the controls.Therefore,we hypothesized that the PI3K-PKB signaling branch affects the food consumption in L.decemlineata through the regulation of expression of sNPF.We detected the temporal and spatial expression profiles of sNPF by real-time PCR,and found that sNPF was mainly expressed in the brain-corpora cardiaca-corpora allata complex and prothoracic gland,and was expressed throughout the life history of L.decemlineata.Knocking down sNPF significantly reduced the food intake by the larvae.In addition,knocking down sNPF did not affect the expression of the LdILP2.Therefore,our results indicate that the PI3K-PKB signaling branch regulates food consumption by control of the expression of sNPF in the L.decemlineata larvae.