| The migratory locust,Locusta migratoria is an important agricultural insect pest,partially due to its high fecundity.Each adult female locust can lay 300-400 eggs.Vitellogenesis is prerequisite to successful egg production and embryonic development after oviposition in insects.During vitellogenesis,the yolk precursor,vitellogenin(Vg)synthesized in the fat body and secreted into hemolymph must pass through the intercellular channels(known as patency)in follicular epithelium to reach maturing oocytes.In many insects including the migratory locust,vitellogenesis is controlled by juvenile hormone(JH),a sesquiterpenoid secreted by corpora allata.JH-induced patency for promoting Vg transportation is a key process in JH-dependent insect reproduction.However,molecular mechanisms of JH action in patency remain to be explored.Using the locust as a model system,this dissertation study was conducted to identify the key factors and signaling pathway associated with zonula adherens and involved in JH-induced patency,by using cellular,molecular and genetic approaches including immunofluorescence,Co-IP,Western blot and RNAi.We expected to reveal the potential targets for sustainable insect pest control.The major findings are listed in the following paragraphs.In locust ovaries,the follicle cells adhere to each other via zonula adherens to form a monolayer follicular epithelium surrounding the oocyte.Immunofluorescence showed that β-catenin,a key protein of zonula adherens was localized in the adhere zone of follicular cells,but absent in the area surrounding patency.Interestingly,the subcellular localization of β-catenin was reversely correlated with patency.When patency enlarged upon JH induction,β-catenin localization was shrunk.These observations indicate that zonula adherens are disassembled when patency is initiated by JH induction.RNAi-mediated knockdown of Par3(partitioning-defective protein 3),Par6 or aPKC(atypical protein kinase C)resulted into zonula adherens break and follicle cell separation,similar to the phenotype caused by β-catenin RNAi.The data suggest that Par3 and its phosphorylation stimulating proteins,Par6 and aPKC function in maintenance of zonula adherens and JH-induced patency.Co-IP showed that JH treatment led to disassociation of β-catenin and Par3,suggesting that JH may promote disassembling of zonula adherens for patency.Phosphorylated aPKC was observed in the area surrounding patency,but not in zonula adherens,implying the involvement of aPKC in patency.When the folliclar epithelium at vitellogenic stage were treated by ACPD,a specific inhibitor of aPKC,JH-induced patency was blocked.Application of ML141,a specific inhibitor of Cdc42 known as the upstream regulator of aPKC and Par6 also inhibited JH-induced patency.These results indicate that Cdc42,aPKC,Par6 and Par3 are likely involved in JH-dependent patency.Western blot showed that JH induced phosphorylation of aPKC and Par3 whereas ACPD or ML141 treatment inhibited JH-stimulated aPKC and Par3 phosphorylation.Taken together,these results indicate that JH is likely to induce the phosphorylation of Par3 via Cdc42-Par6/aPKC signaling pathway,consequently resulting in disassociation of β-catenin and Par3,which led to disassemble of zonula adherens for patency.Treatment of folliclar epithelium at vitellogenic stage with G protein coupled receptor(GPCR)inhibitor suppressed JH-induced patency.In contrast,application of receptor tyrosine kinase(RTK)inhibitors,genistein and Su6668 had no significant effect on JH-induced patency.These data suggest the involvement of GPCR in JH-dependent patency.In summary,this dissertation research demonstrates that β-catenin and zonula adherens play a key role in JH-induced patency.Cdc42,aPKC,Par6 and Par3 transduce JH signaling to stimulate phosphorylation of Par3,leading to disassemble of zonula adherens and consequent patency initiation.The results advance our understanding of molecular mechanism underlying JH-stimulated vitellogenesis in insects. |
PDF Full Text Request