Molecular Mechanism Of Climbing Behavior Of Helicoverpa Armigera Larvae Triggered By HaSNPV

A wide range of parasites manipulate host behaviour as a common strategy to facilitate themselves proliferation and transmission.Lepidoptera larvae infected with baculovirus climbed to the top of plants and then eventually die and liquefy combined to release new progeny virus.This behavioral manipulation of baculovirus was also described as ’tree-top disease’ or ’Wipfelkrankheit’.Recently,tree-top disease induced by baculovirus in their hosts was demonstrated to be egt/EGT-dependent and light-dependent.However,it is unknown which host signaling pathways involved in regulation of baculovirus-induced tree-top disease.In present study,transcriptome and RNAi are employed to identify associated signals and their corresponding functions in HaSNPV-induced climbing behavior in Helicoverpa armigera larvae.The main contents include the following five sections.(1)Tree-top disease induced by HaSNPV was examined under three different test conditions(cotton plants,cotton ropes,and glass tubes)using two doses of HaSNPV.Under all three test conditions,deceased larvae were found at elevated position,indicating that HaSNPV-induced tree-top disease was not depended on the experimental set-up.Larvae infected with higher dose of HaSNPV died at higher position.(2)Deep sequencing technology was employed to screen differentially expressed genes(DEGs)in HaSNPV-infected(T72)compared with mock-infected(C72)larvae,and identified 5972 DEGs which were assigned to 20 main pathways,including 20E and JH signaling,immune and stress responses,and fatty acid metabolic pathways.HaSNPV infection obviously inhibited 20E signaling and promote JH signaling.At last,20 DEGs were randomly selected and verified by qRT-PCR.Expression profiles of selected genes mimicked the changes obtained by RNA-seq with some fold-change discrepancies.(3)Combined hormone treatments and RNAi methods,we demonstrated that 20E signaling negatively controlled HaSNPV-induced tree-top disease by acting on HaSNPV replication,OB production and death height of HaSNPV-infected larvae,while JH signaling antagonized 20E effects and ensured tree-top disease.Western blot and qRT-PCR results demonstrated that 20E induced BrZ2 expression and JH repressed BrZ2 expression at the protein and mRNA levels.After successful knockdown of BrZ2,we observed a significant increasing in HaSNPV replication and OB production,a significantly shorten of LT50 and increasing of the death height of HaSNPV-infected larvae compared with the control groups.(4)Using microarray analysis,we identified 127 significantly differentially expressed host miRNAs after HaSNPV infection.We also predicted that BrZ2 gene was targeted by miR-8 and miR-429 since one potential binding site for these miRNAs was present in the 3’UTR of BrZ2.Subsequent experiments confirmed that miR-8 and miR-429 both negatively regulated the expression of BrZ2,since the absence of miR-8 or miR-429,either by Dicer1 knockdown or specifically using a synthetic inhibitor of miRNAs,increased BrZ2 expresion.In addition,expression of BrZ2 was down-regulated at the transcript and protein levels by miR-8 or miR-429 agomir treatments.These results confirmed that miR-8 and miR-429 directly target BrZ2 and were probably involved in 20E and JH regulated HaSNPV-induced tree-top disease.Further results demonstrated that miR-8 and miR-429 regulated HaSNPV-induced tree-top disease in H.armigera larvae by acting on HaSNPV replication,OB production,and LT50,death height of HaSNPV-infected larvae.Taken together,miR-8 and miR-429 were involved in cross-talk regulation between 20E and JH on HaSNPV-induced tree-top disease in H.armigera larvae via down-regulated BrZ2 expression.(5)As HaTrx2 and HaTrxRl genes were both up-regulated after HaSNPV infection,we further explored their characteristics and functions.Sequence analysis showed that HaTrx2 and HaTrxR1 were both highly conserved and shared high sequence identity with other insect counterparts.The mRNA of HaTrx2 was expressed the highest at 5th instar 96 h and was mainly detected in heads and epidermis.The expression of HaTrxR1 was highly concentrated in 5th instar 72 h and 96 h,and higher in malpighian tube,midgut and hemocyte than other examined tissues.HaTrx2 and HaTrxRl were markedly induced by various types of stress.HaTrx2-or HaTrxR1-knockdown increased ROS production in hemocytes,the lipid damage in HaSNPV-infected H.armigera larvae,and resulted in increased sensitivity to HaSNPV infection,shortened LT50 values.Our findings indicated that HaTrx2 and HaTrxRl contribute to the susceptibility of H.armigera to HaSNPV and also provided the theoretical basis for in-depth study of insect thioredoxin system.