The Study Of Nosema Bombycis Vertical Transmission Mechanism

Nosema bombycis could be transmitted vertically from female to offspring,which is the main reason for its great harm to sericulture.However,the molecular mechanism of its transovarial transmission remains to be elucidated.Our group have conducted a lot of research on mechanism of transovarial transmission of N.bombycis(NbCQ1)in recent years.We found that NbCQ1 could bind to vitellogenin of Bombyx mori(Bm Vg)to infect the ovarioles of Bombyx mori.We further identified three spore wall proteins-NbSWP12,NbSWP26 and NbSWP30 of NbCQ1 could interact with VWD domain of Bm Vg.Yet the function of this combination in the process of infecting ovarioles remains unclear.In addition,NbCQ1 could naturally infect Spodoptera litura,Helicoverpa armigera and other insects.However,there are no information about whether NbCQ1 could transmit vertically in these insects was reported,let alone the functions of NbSWP12,NbSWP26 and NbSWP30.Here,in this study,we further confirmed the combination between NbSWP12,NbSWP26,NbSWP30 and Bm Vg-VWD.On this basis,we analyzed the role of the three spore wall proteins in the transovarial transmission of NbCQ1.Moreover,we identified the characteristics of transovarial transmission of NbCQ1 in S.litura and H.armigera.The main results are as follows:1.Further determination of the combination of NbSWP12,NbSWP26,NbSWP30 and Bm Vg-VWDWe conducted IFA with antibodies of spore wall proteins and Bm Vg to confirm NbSWP12,NbSWP26 and NbSWP30 did bind to Bm Vg at first.We obtained the recombinant proteins of NbSWP12,NbSWP26,NbSWP30 and Bm Vg-VWD in the following.IFA was performed for further determination after incubating spores with r Bm Vg-VWD.Forte Bio Octet was implemented for analyzing the affinity between NbSWP12,NbSWP26,NbSWP30 and Bm Vg-VWD.The results laid a foundation for studying the role of spore wall proteins in the transovarial transmission of NbCQ1.2.The function of NbSWP12,NbSWP26 and NbSWP30 for NbCQ1 in transovarial transmission of Bombyx moriThe recombinant proteins of NbSWP12,NbSWP26 and NbSWP30 were used for competitive combination of Bm Vg-VWD in vivo and vitro,and the infection of ovarioles was significantly decreased.Furthermore,the same is true for antibody blockade experiment to block the spore wall proteins of NbCQ1 in both vivo and vitro.In addition,the load of NbCQ1 in next generation was also decreased.It indicated that NbSWP12,NbSWP26 and NbSWP30 were essential for NbCQ1 to transmit vertically.3.The function of NbSWP12,NbSWP26 and NbSWP30 for NbCQ1 in transovarial transmission of Spodoptera litura and Helicoverpa armigerThe larvae of S.litura and H.armigera were inoculated with NbCQ1.Ovarioles,eggs,offspring were collected from infected female adults for paraffin section.NbCQ1 was found in all collected samples under microscopic observation,which indicated that NbCQ1 could transmit vertically in S.litura and H.armiger.Using the polyclonal antibodies of Sl Vg-VWD and Ha Vg-VWD,the IFA analysis of spores isolated from female pupae showed that Sl Vg and Ha Vg could bind to the surface of spores.Furthermore,IFA and Western blotting were used to detect the protein of NbCQ1 spores isolated from male pupae.It was found that the spore surface could bind to Sl Vg-VWD and Ha Vg-VWD.Then,IFA analysis was carried out on the treated materials with antibodies.It was also found that NbSWP12,NbSWP26 and NbSWP30 could bind to Sl Vg-VWD and Ha Vg-VWD.Finally,we noticed that,the infection of NbCQ1 to the ovarioles of S.litura and H.armiger was significantly reduced when the surface proteins were blocked by antibodies of spore wall proteins.The results showed that NbSWP12,NbSWP26 and NbSWP30 also played an important role in the transovarial transmission of NbCQ1 in S.litura and H.armigera.In conclusion,this study identified the important role of NbSWP12,NbSWP26 and NbSWP30 in the transovarial transmission of NbCQ1 in Bombyx mori,S.litura and H.armigera.NbCQ1 could be transmitted vertically to offspring in S.litura and H.armigera.The results of this study provide a reference for the further analysis of the mechanism of microsporidium transovarial transmission,and provide a potential target for the creation of silkworm resistance materials to pébrine disease.