Characterization Of The Peritrophic Matrix Related Genes In The Western Corn Rootworm (Diabrotica Virgifera Virgifera LeConte)

The western corn rootworm(WCR),Diabrotica virgifera virgifera,is one of the world worst pests of maize.In China,it is in the list of quarantine pests of imported plants.Recently,the WCR has developed resistance to many main insecticides,including BT.The research of the WCR peritrophic matrix can provide a scientific basis for new effective pest control using RNAi methods.As this tissue is absent in higher animals,it could be served as a potential target for biocontrol of pest insects and for developing safe and effective insecticides.In this study,we cloned and sequenced 7 genes cDNAs related to PM.DwCHS2 is the chitin synthase 2 of Diabrotica virgifera virgifera.The gene,DvvCHS2,that contributes to the synthesis of chitin associated with the PM.DvvCHS2 is 4431bp and encode 1476 amino acids.RT-qPCR results showed that the transcripts of DvvCHS2 was specifically expressed in midgut.The DvvCHS2 gene was expressed in all the examined stages,and the highest expression level was found at 3rd larvae and adults.dsCHS2 had a high oral RNAi efficiency to DwCHS2.The RNAi efficiency can be 90%.After fed dsRNA to WCR adults,the expression level of DvvCHS2 dramatically decreased in 1d.However,the adults grew well and no mortality apeared.The susceptibility of carbaryl and chlorpyrifos in WCR adults didn’t change after feeding dsCHS2.DwMgCHT is the midgut specific chitinase of Diabrotica virgifera virgifera.CHT can degrade chitin to low-molecular-weight chitooligosaccharides.DvvMgCHT is 1170bp and encode 389 amino acids.RT-qPCR results showed that the transcripts of DvvMgCHT was specifically expressed in midgut.The DvvMgCHT gene was expressed in all the examined stages,and the highest expression level was found at adults.dsCHT had a high oral RNAi efficiency to DvvMgCHT.After fed dsRNA,the adults grew well and no mortality appeared.However,the susceptibility of chlorpyrifos significantly increased.It suggests that DvvMgCHT may can change the susceptibility of insecticide in WCR.DwPMPC1,DvvPMPC2,DvvPMPC3,DvvPMPC4 and DwPMPS1 are the peritrophic matrix proteins,a.k.a peritrophins,of Diabrotica virgifera virgifera.Peritrophins are the structural components of PM.Through RACE-PCR,3 partial cDNA and 2 full-length cDNA of DvvPMPs were obtained.DvvPMPC2 and DvvPMPC3 are the full-length cDNA with 1320 and 975 bp,and encoded 439 and 324 amino acids,respectively.DvvPMPs were specifically expressed in midgut.The DvvPMPC1-4 were expressed in larvae and adults stages,and the DvvPMPS1 was expressed in larvae,pupae and adults stages.dsPMPs had a high oral RNAi efficiency to DvvPMPC2,DvvPMPC3 and DvvPMPC4.The efficiency can reach 80%and keep high for a couple of days.After fed dsRNA,the adults grew well and no mortality appeared.After feeding dsPMPC4,the susceptibility of chlorpyrifos significantly increased.It suggests that DvvPMPC4 may or may not participate in the insecticide transport between inside the PM and midgut cell in WCR.In this study,we cloned DwCHS2,DvvMgCHT;DvvPMPC1,DvvPMPC1,DvvPMPC3,DvvPMPC4 and DvvPMPSl and get 5 full lenth cDNA of them.WCR is high sensitive to oral RNAi.After silence the DvvMgCHT and DvvPMPC4,the susceptibility of chlorpyrifos changed.All of these studies provide a scientific basis of host induced gene silencing technology and new strategy in IPM.