Study On Molecular Pathway Of Lymantria Dispar Tree-top Disease Caused By Its Viral Pathogen,LdMNPV

In various parasites,host manipulation in protein transcription and expression,cellular metabolism,signal transduction and even behavior is a substantive phenomenon,which has benefited the parasite to increase fecundity and transmission rates.The baculoviruses provide an example of parasite-induced host manipulation,by which infection results in a series of significant behavioral changes in the hosts.In retrospect,the intriguing phenomenon of behavioral change in lepidopterous larvae was first recorded in1891 and was named Tree Top Disease（or“Wipfelkrankheit”）by Hoffmann.Baculoviruses have been shown to perform a number of typical abnormal patterns of behavior,including enhanced locomotory activity（ELA）,extended instar duration and climbing to an elevated location before death,however,the mechanisms of this behavior shifting remain unclear.Based on the results of the infected-larvae’s survival rates,expression levels of Ld MNPV polyhedral matrix protein gene,and behavior change,the transcriptome analysis,metabolome analysis and integrative analysis in four selective time points of Ld MNPV-infection and healthy the 3^rd-instars Lymantria dispar larvae,and the function analysis of relating signal transmission pathways were used to explore the mechanism of“Tree top disease”.The results are as follow:1.Totally,four groups（NB3,CK3,Ld3 and Ld6）from twelve samples of L.dispar larvae transcriptomic sequencing respectively generated functional identity of all Unigenes was investigated basing on information within the GO,Pfam,Swissporot,KOG,egg NOG,Nr,and KEGG databases.The unigenes from each of the two groups were compared to acquire the different expression genes.Taken all reslts of differert databases together,the results show that host gene changes involve many aspects of energy utilization,substance synthesis utilization and intracellular signal transduction.It was shown the significant differences between 3 days post infection（Ld3 group）and 6 days post infection（Ld6group）,indicating differences in host regulation during progeny virions BVs and ODVs production.The cell signal transduction related pathways had changed dramatically after infection.Since the PI3K/AKT signaling pathway is not only necessary for to host cells manipulating,but also deeply involved in insect physiological regulation,it is reasonable to speculate that the fluctuations of the PI3K/AKT signaling pathway may be related to the interaction between virus and host.2.Metabolomic analysis of twenty-four samples from four groups（NB3,CK3,Ld3and Ld6）of L.dispar larvae was performed via UHPLC-QTOF-MS and mapped to KEGG metabolic pathways.The different metabolins analysis between groups showed that the GABAergic synapse,glutarnatergic synapse,ABC-transporters,neuroactive ligand-receptor interaction and phosphatidylinositol signaling system were constant with infection processing,indicating the viral infection may effect on host’s nervous and signal transduction.At the early stage of infection which production was BVs,the nervous system relevant metabolism was impact obviously,and the synapse and neurotransmitter were violent change.At the late stage of infection which production was ODVs,the hormones and relevant signaling pathways were impact acutely.3.Integrative analysis of metabolome and transcriptome screens 52 pathways in the POS mode and 56 pathways in the NEG mode,which the different expression genes and different metabolins were relevanted.Based on the physiological functions of different expression genes and different metabolins between infected and uninfected larvae,it was suggested that the differences of neurotransmitters and cyclohexanol might be more related to the host behavior changes.Especially,in the PI3K/AKT signaling pathway,at the BVs phase,the myo-inositol（MI）was increase and phosphatidyl-1D-myo-inositol（PI）was decrease.The inositol polyphosphate 5-phosphatase（INPP5A）and phosphatidylinositol-4-phosphate 3-kinase（PI3K）gene were upregulated and inositol1,4,5-triphosphate receptor（IP3R）gene was downregulated.And in the ODVs phase,the PI was increase and MI was decrease.Meanwhile,the phosphatidylinositol-3,4,5-trisphosphate3-phosphatase（PTEN）,multiple inositol-polyphosphate phosphatase/2,3-bisphosphoglycerate 3-phosphatase（MINPP1）and IP3R gene were up-regulated,and the PI3K gene was down-regulated.It is means that the dynamic change may relevent to Ld MNPV leading host larvae’extended instar duration and hyperactivity.4.Basing on the results of eighteen genes in PI3K/AKT pathway of fundamental protein analysis and quantitative real-time RT-PCR analysis,remarkable expression pattern of the fifteen genes made it was selected as inferred target.The knowdown rate of ds RNA-pten feeding via oral is 67.43%in 72h post feeding.The results of biological tests after ds RNA-pten interferon showed that at late stage of viral propagate the expression escalation of PTEN gene is necessary for infected larvae dead in high location,which describing as“Tree-top”disease,and also necessary to increasing virion quantity and postponing host death.In summary,we explored that after infecting there had significant changes between Ld MNPV and L.dispar larvae at transcription and metabolism,and uncovered that the PI3K/AKT signaling pathway may play a role in the manipulation of host behavior changes by the baculovirus.It is also showed that the increasing of PTEN gene was important to Ld MNPV leading behavior changes,and the whole PI3K/AKT pathway is deeply involved in hyperactivity during disease progression in Ld MNPV.The above results provided an important basis for elucidating the formation of“Tree top disease”.