The Study On The Relationship Between The Entry Of Yeast-like Symbionts Into Oocyte And The Vitellogenin Absorption Of Oocyte In Brown Planthopper

The rice planthopper,Nilaparvata lugens St?l,is one of important insect pests in Aisa.It causes severe damage to rice not only by sucking phloem sap,spawning in the sheath of rice but also by transmitting viral disease to rice.At present,the disaster caused by BPH is often controlled through spraying chemical pesticides and planting resistant rice varieties.However,it is difficult to manage the disaster because BPH has strong reproduction and adaptability.BPH possesses yeast-like symbionts(YLS),which are related to the resistance of BPH to rice and pesticides.The way the YLS spread between the BPH generations is transovarial transmission.According to the phenomenon that the YLS not enter into the BPH oocytes until the oocytes at vitellogenic stage,this study puts up with a hypothesis that the transovarial transmission of YLS is influenced by the absorption of vitellogenin(Vg)in the oocytes.Three methods were used to verify the hypothesis in this study.First of all,the relationship between the entry of the YLS and the absorption of the Vg in the oocytes in time and space aspects was studied.Furthermore,the Vg expression regulation was inhibited through precocene I(PI)which is an antagonist of juvenile hormone(JH).Third,the expression of Vg and the Vg receptor(VgR)were casted down to depress the synthesis of Vg and the absorption of Vg in the oocytes through RNAi.Here come the results:1.The study on the relation between the entry of YLS into oocytes and the absorption of Vg in the oocytes in time aspect.According to the vitellogenic stage,the BPH follicles are classified into four stages:early vitellogenesis,middle vitellogenesis,late vitellogenesis,final vitellogenesis.The relative transcript level of the Vg and VgR in female BPH adults from the 1st day to the5 th day after emergence was quantitated by fluorescent quantitative PCR(qPCR).The result showed that both of the Vg and VgR tended to be stable after a significant rise on the 2nd day.Considering to the developmental stage of the terminal follicle and the behavior of the YLS enter into oocytes,we found that the YLS entered into oocytes mainly during the middle and late vitellogenesis that coincided to the time the transcript level of the Vg markedly increased.This illustrates the relevance between the entry of the YLS into oocytes and the absorption of oocytes Vg well.Female adults of differentdays after emergence were dissected for statistically analyzing the number of the terminal oocytes at different stage and the follicles with YLS entered.As result,their changes in number were relevant based on relativity analysis.2.The study on the relation between the entry of YLS into oocytes and the absorption of Vg in the oocytes in space aspect.Vitellogenin receptor(VgR)polyclonal antibody was prepared based on the known VgR protein sequence,then the antibody specificity was tested.The distribution of Vg,VgR,lipids as well as Actin in the BPH ovary were observed with the use of immunofluorescence,using VgR and Vg antibodies and other fluorescent dyes.Results are as follows:(1)The specificity of two polyclonal antibodies against VgR was examined by Western-blot and ELISA.One of VgR polyclonal antibody was specific to VgR,as the Western-blot of the female adults and the ovary showed a single line and the change trend in VgR protein content tested by Western-blot and ELISA was corresponding with its relative transcript level tested by qPCR.(2)The channel in the epithelial plug that was differentiated from the follicle cells was the unique structure for the entry of YLS into oocytes.The distribution of Vg or lipids in the terminal oocyte at late vitellogenesis showed polarization because of the convenience the intercellular patency of the follicular cells provided.This regulation of distribution may link to the entry of the YLS into oocytes.Besides,the Actin showed assistant effects during the entry procedure of the YLS into oocytes.3.The validation study on the relation between the entry of YLS into oocytes and the absorption of Vg in the oocytes.(1)Dealt with PI,the BPH females on the 1st day and the 2nd day after emergence were collected to be analyzed.The transcript level of the Vg in PI-dealt BPH significantly was decreased.At the same time,the development of the oocyte was delayed and the number of the YLS entered into the follicle was also reduced,indicating that the Vg expression regulation was inhibited by the PI and then influenced the entry of the YLS into the oocytes.(2)BPH females emerged in 12 hours were injected with dsVg,in comparison to dsGFP,the Vg relative expression level dropped more than 95 %,the development of ovary were inhibited and the terminal follicles remained before final vitellogenesis witha few YLS entered on the 3rd day after emergence.The results indicate that the injection of dsVg is failed to stop but to some extent inhibited the absorption of Vg into oocytes leading to the entry of a few YLS.Injected with dsVgR,the terminal follicles of BPH remained early or before vitellogenesis without the entry of the YLS into oocytes on the3 rd day after emergence.As VgR is the Vg receptor on the follicle cells,the RNAi of VgR promised to work better,compared with the RNAi of Vg.