RNA Inteference Of Amino Acid Absorption And Transport Related Genes Disturbs Insulin Signaling Pathway 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 this study,bHLH members,genes related to insulin/insulin-like growth factor signaling pathway(IIS),a vacuolar ATPase subunit E gene and a nutrient amino acid transporter gene were identified.We knocked down LdILP2,LdATPaseE and LdNAT1 by RNAi,observed the influence on larvae ecdysis,and clarified molecular mechanisms underlining the phenotypes.The main results were shown as fellows.1.Constructing and characterization of insulin/insulin-like growth factor signaling pathway(IIS)Based on the transcriptome and the genome of the Colorado potato beetle Leptinotarsa decemlineata,49 bHLH members were identified in the most important pest in potato.All LdbHLH members were defined by their names and families according to various phylogenetic analyses with bHLH homologues of D.melanogaster,A.mellifera,B.mori and T.castaneum.Among these LdbHLHs,17,10,10,1,10 and 1 members belonged to A,B,C,D,E and F high-order groups,respectively.Moreover,58 genes related to insulin/insulin-like growth factor signaling pathway(IIS)were cloned.Among them 53 had complete ORF.IIS pathway was described accordingly.Furthermore,the expression patterns of 5 LdILPs(LdILP1a,LdILP1b,LdILP1c,LdILP2 and LdILP4)were measured at different developing stages and in various tissues.RNAi-mediated knockdown of LdILP2 significantly lowered larval fresh weight,and impaired pupation and adult emergence.Silencing LdILP2 disturbed IIS signaling,28 related genes were downregulated,whereas 8 genes were upregulated.In addition,knockdown of LdILP2 inhibited JH and 20E signaling pathways.2.RNA-mediated knockdown of LdATPaseE impaired larval ecdysisHere we cloned and characterized a 1041-bp full-length vATPase subunit E cDNA(named as LdATPaseE)that encoded a 226-amino acid protein in Leptinotarsa decemlineata.At the first,second and third instar stages,the expression levels of LdATPaseE were lower just before the molt.At the final instar stage,the LdATPaseE trough occurred 96 hours after ecdysis It was highly expressed in ileum and rectum,moderately expressed in Malpighian tubules,midgut and foregut,and lowly expressed in fat body,ventral ganglion,epidermis and haemocytes in the fourth instars.It is suggested that a clear relationship between the levels of circulating 20-hydroxyecdysone(20E)/juvenile hormone(NIJHOUT and WILLIAMS)and the expression levels of LdATPaseE.In vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide repressed LdATPaseE expression,whereas a decrease in 20E by RNA interference of an ecdysteroidogenesis gene LdSHD and a disruption of 20E signaling by RNAi of a 20E response gene LdFTZ-F1 activated the expression.Thus,LdATPaseE responded to 20E signaling.Ingestion of dsLdATPaseE-1 and dsLdATPaseE-2 by the second-instar larvae successfully knocked down the target gene,inhibited larvae growth,and increased larval mortality.Ingestion of dsLdATPaseE-1 and dsLdATPaseE-2 at very low concentration by the third-and fourth-instar larvae also successfully knocked down the target gene,inhibited the larvae growth,and significantly impaired pupation.Moreover,silencing LdATPaseE significantly enhanced the expresion of both LdInR and Ld4EBP,inhibited the expression a 20E biosynthesis enzyme gene,lowered 20E titer,and downregulated the expression of a 20E response gene.Knocking down LdATPaseE also inhibited the expression a JH biosynthesis enzyme gene,lowered JH titer,and downregulated the expression of an early JH response gene.Therefore,knockdown of LdATPaseE inhibited IIS pathway,lowered 20E and JH titers,disturbed 20E and JH signaling pathways,and subsequently affeted larval grwoth and impaired development.3.Knockdown of a nutrient amino acid transporter gene LdNATl impairs pupationInsect midgut cells actively absorb essential amino acids by the SoLute Carrier family(SLC)Nutrient Amino acid Transporters(NAT).In the present paper,a putative LdNAT1 belonging to SLC6 family was cloned from Leptinotarsa decemlineata.LdNAT1 mRNA levels were higher in the midgut,moderate in the foregut,hindgut and Malpighian tubules,and lower in other tested tissues.Its transcripts were higher right after the molt,and were lower just before the molt,suggesting a clear relationship between the levels of circulating 20-hydroxyecdysone(20E)/juvenile hormone(NIJHOUT and WILLIAMS)and the expression levels of LdNAT1.In vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide repressed LdNATl expression,whereas a decrease in 20E by RNA interference of an ecdysteroidogenesis gene LdSHD and a disruption of 20E signaling by RNAi of a 20E response gene LdFTZ-F1 activated the expression.Conversely,JH,a JH analog pyriproxyfen and an increase in JH by RNAi of an allatostatin gene LdAS-C upregulated LdNAT1 expression,whereas a decrease in JH by RNAi of a synthesis gene LdJHAMT reduced the expression.Thus,LdNAT1 responded to both 20E and JH signaling.Moreover,comparison of free amino acid content from the bodies and the feces indicated that knockdown of LdNATl affected the absorption of cysteine,histidine,isoleucine,leucine,methionine,phenylalanine and serine by the larval gut.As a result,silencing of LdNAT1 inhibited insulin pathway,modulated 20E and JH titers,disrupted 20E and JH signaling,afftected larval growth and impaired pupation.Therefore,LdNAT1 was a functional NAT-SLC6 member in L.decemlineata.