Cloning And Functional Characterization Of Trehalose Metabolism And Chitin Biosynthesis-related Genes 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.Identification of the genes involved in trehalose metabolism and chitin biosynthesis may facilitate to develop potential control strategies.In the present paper,thirty-two carbohydrade and lipid metabolism-related genes were cloned.Using RNA intererence(RNAi),LdTPS,LdTRE,LdUAP and LdChS were successfully knocked down.The influence of RNAi on larvae and female adults were observed.The roles of these genes played in the metabolism of trehalose and the biosynthesis of chitin were clarified.The main results were shown as fellows.1.Molecular cloning of carbohydrade and lipid metabolism-related genes and transcription response of LdTPS,LdTRE,LdUAP and LdChS genes to hormonal stimulationBased on L.decemlineata transcriptome and genome data,thirty-two full-length cDNAs belonging to carbohydrade and lipid metabolism-related genes were cloned.Among these genes,twenty-eight encoded enzymes involved in trehalose metabolism,chitin synthesis,glycogen synthesis and degradation,glycolysis,citrate cycle,pentose phosphate and gluconeogenesis pathways.The other five genes were responsible for fatty acid biosynthesis and lipid degradation.Temporal expression patterns of nine trehalose metabolism and chitin synthesis-related genes revealed that their expression tops or bottoms were respectively correlated with circulating 20-hydroxyecdysone(20E)and juvenile hormone(JH).In vitro midgut culture or in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide(Hal)enhanced the expression of trehalose-6-phosphate synthase gene(LdTPS),trehalase(LdTRE),Uridine diphosphate-N-acetylglucosamine-pyrophosphorylase(LdUAP)and chitin synthase(LdChS)in larvae.Conversely,a decrease in 20E or disruption of 20E signaling by knockdown of LdSHD,LdTorso,LdEcR,LdE75,LdHR3 or LdFTZ-F1 repressed their expression.Thus,20E pulses activate the transcription of LdTPS,LdTRE,LdUAP and LdChS in a 20E signaling dependent manner.Similarly,in vitro culture or in vivo bioassay showed that exogenous JH or JH analogs(methoprene and pyriproxyfen)or RNAi of LdAS-C activated LdTPS,LdTRE,LdUAP2,and LdChS,and repressed LdUAP 1 expression.In contrast,a decrease in JH by silencing of LdJHAMT downregulated LdTPS,LdTRE,LdUAP2,and LdChS,and increase LdUAP1 expression.The transcription responses of LdTPS,LdTRE,LdUAP and LdChS to 20E and JH signaling pathways indicate their critical physiological roles in the larval development and adult reproduction in L.decemlineata.2.Roles of trehalose in the larvae revealed by RNAi of trehalose-6-phosphate synthase and trehalase genesTrehalose is proposed to serve multiple physiological roles in insects.However,its importance remains largely unconfirmed.In this chapter,we knocked down either a trehalose biosynthesis gene LdTPS or each of three degradation genes(soluble LdTRE1a,LdTRE1b or membrane-bound LdTRE2)by RNAi.Knockdown of LdTPS decreased trehalose content and caused larval and pupal lethality.The LdTPS RNAi survivors consumed a greater amount of foliage,obtained a heavier body mass,accumulated more glycogen,lipid and proline,and had a smaller amount of chitin compared with the controls.Ingestion of trehalose but not glucose rescued the food consumption increase and larval mass rise,increased survivorship,and recovered glycogen,lipid and chitin to the normal levels.In contrast,silencing of LdTRE1a increased trehalose content and resulted in larval and pupal lethality.The surviving LdTRE1a RNAi hypomorphs fed a smaller quantity of food,had a lighter body weight,depleted lipid and several glucogenic amino acids,and contained a smaller amount of chitin.Neither trehalose nor glucose ingestion rescued these LdTREla RNAi defects.Silencing of LdTRE1b caused little effects.Knockdown of LdTRE2 caused larval death,increased trehalose contents in several tissues and diminished glycogen in the brain-corpora cardiaca-corpora allata complex(BCC).Feeding glucose but not trehalose partially rescued the high mortality rate and recovered glycogen content in the BCC.It seems that trehalose is involved in feeding regulation,sugar absorption,brain energy supply and chitin biosynthesis in L.decemlineata larvae.3.LdUAP and LdChS encode functional enzymes involved in chitin synthesisUAP and ChS are involved in the biosynthesis of chitin,an essential component of the epidermal cuticle and midgut peritrophic matrix(PM)in insects.two putative LdUAP(LdUAP1 and LdUAP2)and three LdChS(LdChSAa,LdChSAb and LdChSB)genes were cloned in Leptinotarsa decemlineata.LdChSAa and LdChSAb were two splicing variants of LdChSA gene.LdChSAa,LdChSAb and LdUAP1 were highly expressed in ectodermally-derived epidermal cells forming epidermis,trachea,foregut and hindgut,whereas LdUAP1 and LdChSB was mainly transcribed in midgut cells.Knockdown of LdUAP1 or LdChSA reduced chitin contents in whole larvae and integument samples,made the tracheal taenidia slender,impaired larval-larval molt,larval-pupal ecdysis and adult emergence.In contrast,silencing of LdUAP2 or LdChSB significantly reduced foliage consumption,decreased chitin content in midgut samples,damaged PM,and retarded larval growth.The resulting larvae had lighter fresh weights,smaller body sizes and depleted fat body.As a result,the development was arrested.Knock down either LdChSAa or LdChSA has similar effect.In contrast,knockdown of LdChSAb mainly affected pupal-adult molting.Combined knockdown of LdUAP1 and LdUAP2 caused an additive negative effect.Our data reveal that LdUAP1 and LdChSA specially catalyze the biosynthesis of chitin in the epidermal cuticle,whereas LdUAP2 and LdChSB catalyze the production of chitin in PM.Moreover,the results suggest that LdUAPs and LdChSs are among the most attractive candidate genes for an RNAi-based control strategy against L.decemlineata.4.Knockdown of trehalose metabolism and chitin synthesis-related genes affects oogenesis in the femalesTrehalose and chitin have been documented to be involved in oogenesis in a few insect species.The temporal expression profiles of the eight trehalose synthesis and degradation,chitin synthesis genes were determined in the day 0 through day 10 adults.LdTPS,LdTRE1a,LdTRE2,LdUAP1 and LdUAP2 transcripts peaked at the day 3-5 and day 9-10 females.In contrast,LdChSA was highly expressed in day 0 and then gradually lowered.LdChSB transcript reached its peak on day 3-5 females.Tissue expression analysis revealed that LdTPS,LdTRE1a,LdTRE1b,LdTRE2,LdChSA and LdChSB was specific expressed in fat body,epidermis and gut,testis,ovaries,epidermis and gut,respectively.The highest expression of LdUAPl was found in ovaries;its mRNA level was also high in epidermis and gut.LdUAP2 mainly expressed in gut,epidermis and ovaries.Knockdown of either of the four trehalose metabolism genes(LdTPS,LdTRE1a,LdTRE1b,LdTRE2)significantly affected trehalose contents and caused adult lethality.The RNAi survivors obtained lighter fresh weight,and deposited fewer eggs.Dissection revealed that oogenesis wad delayed in the LdTPS,LdTRE1a and LdTRE2 RNAi females were.The size of the ovary was reduced.There were a greater number of atresic oocytes and a smaller number of chorionated oocytes,compared with the control.Similarly,silencing of LdUAP2,LdChSA and LdChSB declined female fresh weight.Knockdown of LdUAP2,LdChSA and LdChSB caused adult lethality and failure of oviposition.Moreover,the size of the ovary was reduced,and the development of oocytes was disrupted.Further analysis showed that abnormal sugar contents in haemolymph disrupted ILP signaling.As a results,the transcripton and transaction of vitellogenin genes were reduced.Thus,it seems that normal metabolism of trehalose and chitin are critical for adult growth,oogenesis,and the maintain of ovary structure in L.decemlineata females.