RNA Inteference-mediated Knockdown Of Nuclear Receptor Gene E75 And HR3 On Pupation In Leptinotarsa Decemlineata

Leptinotarsa decemlineata is the most destructive pest of potato,and is a major quarantine pest in China.The outbreak of the beetles results in large quantity of yield loss.Exploretion of the molecular mechanisms underlining metamorphsis may facilitate to develop potential control strategies.In the present paper,we cloned and characterized two RNA interference core genes,Dicer2(Dcr2)and Argonaute2(Ago2).We identified all members belonging to the nuclear receptor family.Finally,we explored the molecular mechanisms of E75 and HR3 to regulate larval-pupal metamorphsis.The main results were shown as fellows.1.Characterization of Dicer2(Dcr2)and Argonaute2(Ago2)To study the role of Dicer2 and Argonaute2 genes in RNAi efficiency,we identified LdDcr2a,LdDcr2b,LdAgo2a and LdAgo2b.Their expression levels were higher in young larvae than those in old ones.Exposed to dsegfp for 6 h significantly elevated LdDcr2a,LdDcr2b,LdAgo2a and LdAgo2b mRNA levels in the first-,second-,third-and fourth-instar larvae.When the exposure periods were extended,however,the expression levels were gradually reduced.Continuous exposure for 72 h significantly repressed the expression of LdAgo2a and LdAgo2b in the first,second and third larval instars,and the four genes in final instars.Moreover,we found that dsLdSAHase-caused LdSAHase suppressions and larval mortalities were influenced by previous dsegfp exposure:12 h of previous exposure increased LdSAHase silencing and mortality of the final instar larvae,whereas 72 h of exposure reduced LdSAHase silencing and mortality.Thus,it seems the activities of core RNAi-machinery proteins affect RNAi efficiency in L.decemlineata.2.Identification of the nuclear receptor membersBased on the transcriptome and the genome data,21 nuclear receptor(NR)members were identified.All NR members were defined by their names and subfamilies according to various phylogenetic analyses with NR homologues of D.melanogaster and T.castaneum.Among these NRs,2,5,9,1,1,2 and 1 members belonged to NRO,NR1,NR2,NR3,NR4,NR5 and NR6 subfamilies.Two members in NRO subfamily were defined as NR0A2(LdKNRL)and NR0A3(LdEagle).Five in NR1 subfamily were named as NR1D3(LdE75),NR1E1(LdE78),NR1F4(LdHR3),NR1H1(LdEcR)and NR1J1(LdHR96).NR2 subfamily has the most members,a total of nine were found.They were NR2A4(LdHNF4),NR2B4(LdUSP),NR2D1(LdHR78),NR2E2(LdTLL),NR2E3(LdHR51),NR2E4(LdDSF),NR2E5(LdHR83),NR2E6b(LdNameless)and NR2F3(LdSVP).Either NR3 or NR4 subfamily has only one member,i.e.,NR3B4(LdERR)and NR4A4(LdHR38).Two members in NR5 subfamily were designated NR5A3(LdFTZ-F1)and NR5B1(LdHR39).The one in NR6 subfamily was defined as NR6A1(LdHR4).3.Nuclear receptor E75 is required for the larval-pupal metamorphosisDuring the final-instar larvae,a pulse of 20-hydroxyecdysone(20E)and a drop in juvenile hormone(JH)trigger larval-pupal metamorphosis.In this study,two LdE75 cDNAs(LdE75A and LdE75B)were cloned from Leptinotarsa decemlineata.Both LdE75 isoforms were highly expressed just before or right after each molt.Within the fourth larval instar,both showed a smaller rise and a bigger peak 40 and 80 hours after ecdysis.In vitro midgut culture and in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide(Hal)enhanced LdE75A and LdE75B expression in the final larval instar.Conversely,a decrease in 20E by feeding a double-stranded RNA(dsRNA)against an ecdysteroidogenesis gene LdSHD repressed the expression.Moreover,Hal rescued the expression levels in the LdSHD-silenced larvae.Thus,20E pulses activate the transcription of LdE75s.Furthermore,ingesting dsE75-1 and dsE75-2 from a common fragment of the two isoforms successfully knocked down both LdE75s,and caused development arrest.Finally,knocking down LdE75s significantly repressed the transcription of three ecdysteroidogenesis genes,lowered 20E titer,and reduced the expression of two 20E-response genes.Silencing LdE75s also induced the expression of a JH biosynthesis gene,increased JH titer,and activated the transcription of a JH early-inducible gene.Thus,LdE75s are required for larval-pupal metamorphosis mainly by modulating 20E and JH titers and mediating their signaling pathways.4.Functions of nuclear receptor HR3 during larval-pupal molting revealed by in vivo RNA interferenceWe characterized a putative LdHR3 gene,an early-late 20E-response gene upstream of LdFTZ-F1.Within the first,second and third larval instars,three expression peaks of LdHR3 occurred just before the molt.In the fourth(final)larval instar 80 hours after ecdysis and prepupal stage 3 days after buring into soil,two LdHR3 peaks occurred.The LdHR3 expression peaks coincide with the peaks of circulating 20E level.In vitro midgut culture and in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide(Hal)enhanced LdHR3 expression in the final larval instars.Conversely,a decrease in 20E by feeding a double-stranded RNA(dsRNA)against an ecdysteroidogenesis gene Ldshd repressed the expression.Moreover,Hal rescued the transcript levels in the Ldshd-silenced larvae.Thus,20E peaks activate the expression of LdHR3.Furthermore,ingesting dsRNA against LdHR3 successfully knocked down the target gene,and impaired pupation.Finally,knocking down LdHR3 upregulated the transcription of three ecdysteroidogenesis genes(Ldphm,Lddib and Ldshd),increased 20E titer,and activated the expression of two 20E-response genes(LdEcR and LdFTZ-Fl).However,silencing LdHR3 did not affect JH titer and JH signaling.Thus,LdHR3 functions in regulation of pupation in the Colorado potato beetle.