The Study On The Cytomorphology Of Insect Vectors Infected By Three Species Of Rice Viruses

Most rice viruses in Reoviridae family are transmitted by Hemiptera,Delphacidae or Cicadellidae insects in a persistent-propagative manner,among which Southern rice black-streaked dwarf virus is transmitted by its competent vector White-backed planthopper(WBPH;Sogatella furcifera).The incompetent vector of SRBSDV,Small brown planthoppers(SBPH;Laodelphax striatellus)can also carry this virus,but incapable to transmit it.Rice gall dwarf virus(RGDV)and Rice dwarf virus(RDV)are transmitted by leafhopper Recilia dorsalia and Nephotettix cincticeps,respectively.When viruses are ingested by insects from diseased rice,they firstly enter and replicate in the epithelial cells of the alimentary canal and then transmitted into the haemocoele of insect vectors.Meanwhile,they need to overcome tissue barriers and immunity barriers during virus transmission.In this study,immunofluorescence microscopy and transmission electron microscopy(TEM)were exploited to describe the histological and ultrastructural changes of insect alimentary canal caused by viral replication in the midguts.Moreover,in order to observe the abnormality of alimentary canal,RNAi assay was applied to knockdown the expression of Dicer2(Dcr2)in the insects.The results were as below:1)Non-viruliferous alimentary canals of planthoppers and leafhoppers were observed by TEM.The alimentary canals of both planthoppers were long,hollow and tubular,similar in histology and ultrastructure.One side of the alimentary canal was connected to the mouthpart and another was connected to the anus.The midgut mainly consisted of a monolayer of epithelial cells,with the microvilli on the lumen side and basement membrane on the hemocoel side.The basement membrane was covered with muscle fiers.Plenty of mitochodria were distributed in the epithelial cell of the midgut.Moreover,the ultrastructure of the midgut in leafhopper was similar to the planthopper.The end of esophagus was connected to the filter chamber followed by the midgut shaped like the letter "U" both sides of which were connected to the filter chamber.So the whole alimentary canal forms a loop structure.The membrane structure covered on the microvilli was perimicrovillar membrane.2)Insect vectors infected with viruses were tested under natural condition.We dissected the midguts at the 5th day after fed on diseased plants then observed the viral accumulation and transmission in vitro.The results showed that rice viruses in the competence vector were able to reach the muscle layers without much propagation in the epithelial cells.However,SRBSDV could not disseminate into the muscle layers of its incompetent vector SBPH and was restricted in the epithelial cells.Plant virus would not injure the insect vectors when they propagated at a lower level.So we speculated that there must be a delicate balance between the viral propagation and the health of insect vectors.3)In this study,the viral propagation can be modulated by RNAi technology.The expression of the core component Dcr2 of siRNA pathway was knockdown in vivo.The results showed that the propagation ability of these viruses was increased obviously.Meanwhile,Lesions were observed in the epithelial cells of midgut which were full of viruses.The systemic infection and propagation of SRBSDV in the midgut of WBPH caused severe cytopathological changes such as vacuolization in the epithelium,dispersion of cytoplasm,rupturing and damage of microvilli and peritrophic membrane.The similar situation occurred in the alimentary canal of RGDV infected R.dorsalia.On the contrary,the accumulation of RDV in its competent vector N.cincticeps was just increased slightly after injected with dsRNA,therefore no obvious cytopathological changes occurred.In its incompetence vector,SRBSDV accumulated at a high level in the midgut of SBPH and some viral particles could disseminate from midgut epithelium into the muscle layers through a tubular structure.Most viruses were limited in the epithelial cells and cytopathological changes in each viruliferous epithelial cells were obvious,such as swelling of cells,high electron-dense,rupturing and damage of microvilli and peritrophic membrane.There were significant ultrastructural differences between the viruliferous epithelial cells and the adjacent cells.In summary,we used immunofluorescence microscopy,TEM and RNAi technology to confirm that(a)the normal accumulation of the 3 rice viruses would not injure the ultrastructure of the alimentary canal of their insect vectors and it was modulated by the antiviral immunity pathway and various insect barriers.(b)Dcr2,the core component of insect vector siRNA pathway,played an important role in controlling viral accumulation and protecting insects.The results in this study not only provided the convenience for exploring the interactions between virus and its insect vector,but also put forward new insights for controlling the arbovirus and pest.