Exploration Of Taiman-mediated 20-Hydroxyecdysone Signaling In Leptinotarsa Decemlineata Larvae

The Colorado potato beetle Leptinotarsa decemlineata is an important quarantine pest in China.It seriously threatens the production of potato.The beetle is a holometabolous insect,and the larval-pupal-adult transition is tightly regulated by 20-hydroxyecdysone(20E)and juvenile hormone(JH).In the process of metamorphosis,both the cuticle and internal tissues and organs undergo dramatical changes.Therefore,clarifying the multiple branches of hormone signaling and the role of 20E in the regulation of metamorphosis have important theoretical significance and potential application value.Insect Taiman binds to Methoprene-tolerant to form a heterodimeric complex,mediating juvenile hormone(JH)signaling to regulate larval development and to prevent premature metamorphosis.Taiman also acts as a steroid receptor coactivator of 20-hydroxyecdysone(20E)receptor heterodimer,ecdysone receptor(EcR)and Ultraspiracle(USP),to participate in insect reproduction.In holometabolous insects,however,whether Taiman functions as the coactivator of EcR/USP to transduce 20E message during larval-pupal transition is unknown.Here we focus on the role of Taiman and its downstream transcription factors HR4 and E74 in the growth and metamorphosis in the larvae.The main results are as follows.1.Taiman impairs larval developmentFive isoforms and their exons composition of Taiman,a transcription factor belonging to the bHLH-PAS family were identified in L.decemlineata.The N terminal of Taiman has a conserved bHLH-PAS domain and in the middle region has a LxxLL motif.Taiman was expressed in various larval tissues and at different larval developing stages,which indicates the importance of Taiman in larval growth and development.Therefore,we designed and synthesized dsTai in the common sequence of the five LdTai isoforms.We found that continuous ingestion of dsTai dipped the potato foliage for three days by third(penultimate)-instar larvae caused approximately 20%larval mortality and 80%pupation failure.The larval lethality resulted from abnormal cuticle and tracheae shedding.The exuviae fragments were attached to the appendages such as legs and mouthparts,which subsequently reduced foliage consumption and nutrient absorption,and depleted lipid stores.In dsTai larvae,pupation failure was due to disturbed JH and 20E signals,leading to developmental and nutritional balance disorders,including inhibition of trehalose metabolism,reduction of chitin content,and over-accumulation of glycogen and lipids.Furthermore,knockdown of LdTai caused similar larval lethality and pupation impairment in second larval instars.It seems that LdTai mediates JH and 20E pathways,which are crucial for the normal growth and molting of larvae in L.decemlineata.2.Molecular mechanism of Taiman in larval-pupal transitionIn this chapter,we found that the LdTai mRNA levels were positively correlated with circulating JH and 20E titers in L.decemlineata.External application of JH or 20E significantly induced the transcription of LdTai.Knockdown of LdTai at the fourth(final)instar stage repressed both JH and 20E signals.Although the titer of JH and 20E in dsTai fed larvae was increased,the expression of its downstream signal transduction factors were significantly inhibited.Due to the inhibition of JH signal transduction,the larval growth was significantly inhibited and the development period was significantly shortened,compared with those in the control group.As the 20E signal transduction was inhibited,all the LdTai RNAi larvae failed to molt and eventually died.Under the apolysed larval cuticle,the LdTai RNAi prepupae possessed pupal thorax.In addition to this,the processes of tracheal ecdysis and fat body remodeling were uncompleted.Neither JH nor 20E rescued the aforementioned defectives in LdTai RNAi larvae.We accordingly propose that LdTai can simultaneously mediate JH and 20E signaling during larva to pupa metamorphosis.In JH signal transduction,LdTai and LdMet form heterodimers to regulate the transcription of downstream genes.In 20E signal transduction,LdTai acts as a co-activator of the 20E receptor complex EcR/USP,and is involved in the regulation of expression of downstream transcription factors such as E74 and HR4.3.LdHR4 tunes ecdysteroidogenesis and mediates 20-hydroxyecdysone signaling during larval-pupal metamorphosisHere we characterized LdHR4 and found two splicing variants,LdHR4X1 and LdHR4X2,in L.decemlineata.LdHR4X1 was highly expressed in the prothoracic gland and epidermis while LdHR4X2 was abundantly transcribed in the nervous system.The time expression of the two LdHR4 isoforms were consistent with the peak of 20E in hemolymph circulation.In vivo results showed that PTTH inhibited the expression of LdHR4 in the PG,while 20E induced the transcription of LdHR4.RNA interference of LdHR4 at the fourth(final)larval instar enhanced the expression of two ecdysteroidogenesis genes(LdDIB and LdSHD),increased 20E titer,upregulated transcription of four 20E-response genes(LdEcR-A,LdEcR-B,LdE75 and LdHR3),and reduced the mRNA level of LdFTZ-F1.As a result,the fourth-instar LdHR4 RNAi larvae increased larval mortality exhibited,accelerated development and reduced body weight.Moreover,the larvae could enter the soil,but they could not complete the metamorphosis from larva to pupa,and eventually all died.Feeding of pyriproxyfen(a mimic of juvenile hormone)restored the normal course of ecdysteroidogenesis,duration of larval development,and body weight in fourth-instar LdHR4 RNAi larvae.The treatment partially rescued the larval mortality but not the failure to pupate and the declining expression of LdFTZ-F1.It is believed that LdHR4 plays a dual role in larval-pupal metamorphosis.One is involved in the synthesis of 20E in the PG and the other is involved in the signal transduction from larva to pupa as a signal transduction factor in the peripheral tissues.4.Requirement of LdE74 for larval-pupal metamorphosis and appendage growthTwo isoforms(LdE74A and LdE74B)of LdE74 gene were identified in L.decemlineata.Their protein shared a common C-terminal ETS DNA-binding domain but differ in their N-terminal.During the larval development stage,the mRNA transcript levels of the two LdE74 isoforms were correlated with circulating 20E titres.In vitro midgut culture and in vivo dietary supplementation with 20E revealed that the presence of 20E induced expression peaks of both LdE74A and LdE74B,with similar patterns observed for the two isoforms.Moreover,the mRNA transcript levels of both LdE74A and LdE74B isoforms were significantly downregulated in the LdEcR RNAi specimens,but not in the LdE75 RNAi beetles.We designed dsRNA in their common area to knock down all the two isoforms.The results showed that silencing LdE74 in the fourth(final)larvae caused failure of ecdysis.Most of the LdE74 RNAi beetles remained as prepupae.The LdE74 RNAi prepupae exhibited adult character-like forms underneath after removal of the apolysed larval cuticle.Their appendages such as antennae,legs and wings were shorter than those in control larvae.Only a few LdE74 RNAi larvae finally became deformed pupae,with shortened antennae and legs.Furthermore,ingestion of 20E reduced the larval fresh weights and shortened the larval development period,irrespective of knockdown of LdE74 or not.RNAi of LdE74 did not affect 20E-induced expression of the E75-HR3-FTZ-F1 transcriptional cascade.It is accordingly hypothesized that LdE74 is required for larval-pupal metamorphosis and appendage growth in L.decemlineata.Meanwhile,it seems that LdE74 mediates 20E signalling independent of the E75-HR3-FTZ-F1 transcription pathway.5.Taiman and HR4 are required for the gut-clearing before pupationIn many kinds of insects,wandering larvae stop feeding,and purge their gut contents before pupation.In this chapter,we found the LdTai or LdHR4 knockdown larvae could not complete the gut-clearing before pupation.The intestinal tracts of the treated larvae were filled with heavily-melanized food residues.In the previous chapter,we confirmed that LdTai of L.decemlineata upregulated the expression of LdHR4,so we focused on the gut-clearing mechanism of LdHR4.Our comparative studies revealed that knockdown of LdHR3 affected metamorphosis from larva to pupa,but did not influence the gut-clearing.When we interfere with LdHR4 and LdHR3 at the same time,the metamorphosis and gut-clearing were affected.Knockdown of LdHR4 repressed transcription of LdUAP2.As a result,the chitin content in the midgut was reduced and the structure of the peritrophic membrane was impaired.However,RNA interference with LdHR3 did not affect the content of chitin in the midgut and the structure of the peritoneal membrane.Quantitative detection showed that the genes involved in the melanogenesis pathway were overexpression and the melanization was enhanced in the LdHR4 RNAi larvae gut.Similarly,when TcHR4 was interfered in the middle of the final instar,the larvae could not finish gut-clearing,and the foregut and the hindgut were darkened.Therefore,20E main signal pathway EcR/USP-HR3-FTZ-F1 is not necessary to initiate gut-clearing.Conversely,Tai and HR4 regulate the gut-clearing before pupation by affecting the integrity of the midgut peritrophic matrix and melanization of food residues in L.decemlineata larvae.