Establishment Of An In Vitro System For Patency Study And Identification Of Mediators Involved In Patency Regulation In Locusta Migratoria

The migratory locust,is an important agricultural insect pest in China,partially due to its high fecundity.During insect egg maturation,a large amount of yolk proteins are required to be deposited into the developing oocytes,which provide nutrients for embryonic development after oviposition.Vitellogenesis is prerequisite to egg production.In locusts,vitellogenin(Vg)is synthesized in the fat body,transported by the hemolymph,and taken up by developing oocytes.Insect oocytes are surrounded by an array of follicle cells constituting the follicular epithelium.To reach the surface of oocytes,hemolymph.Vg needs to pass through the intercellular spaces(named patency)of follicle cells,then deposited into the maturing oocytes by receptor-mediated endocytosis.Previous studies have shown that juvenile hormone(JH)controls the openness of patency.However,an effective in vivo or in vitro model system to study the regulatory mechanisms of patency has been lacking,and the central mediators in JH-regulated patency functioning remains unrevealed.The migratory locust is an ideal model to elucidate the molecular mechanism of JH regulation in patency functioning,as JH control locust vitellogenesis and patency opening.Moreover,the patency in follicular epithelium of locusts responds JH quickly.This dissertation research was therefore aimed to:1)determine the best staining dyes for patency observation in vivo and in vitro;2)establish approaches of live imaging for patency examination in vivo and in vitro and for exploring the mechanisms of JH regulation in patency functioning;3)identify the members of G-protein coupled receptor(GPCR)involved in JH membrane signal transduction and patency regulation.The results showed that CellMask Orange Plasma Membrane Stain was an ideal dye for patency staining and live imaging.By using this staining approach,we examined the developmental profiles of patency development in the first gonadotrophic cycle of adult female locusts.The patency was not seen when the primary oocytes was<1.5 mm in length,corresponding to the previtellogenetic stage.During the vitellogenic phase,the patency was observed when the primary oocytes were>1.5mm,then enlarged gradually and reached the peak when the primary oocytes were at 5.5 mm.However,the patency was undetectable after the primary oocyte were>6.0 mm.It appeared that the patency was opened during the entire vitellogen stage.To establish an in vitro JH-induced model,we employed JH III and a potent JH homolog(JHA),methoprene to treat the cultured follicle epithelia and induce the patency.We demonstrated that follicle cells were shrunk and patency was enlarged within 1 hour after JH and JHA treatment at a dose of 10~-88 M.These observations would be helpful for in establishment of platform in exploring the molecular basis of JH induction patency and JH membrane signaling.GPCR family members are candidates for transducing JH signaling to induce the patency.In this study,12 genes coding for GPCRs were selected to investigate their roles in vitellogenesis via RNAi.We found that knockdown of orphan receptor(OrR),cardioacceleratory peptide receptor(CCAPR),methuselah-like2(Mthl2),parathyroid hormone/parathyroid hormone-related peptide receptor(PTHR)or tyramine receptor 2(TyR2),resulted in significantly inhibited ovarian growth and oocyte maturation.Interestingly,our western blots showed that depletion of OrR or CCAPR led to increased levels of Vg proteins in the fat body and hemolymph but decreased levels of Vg protein in the ovary.These results indicate that the arrested ovarian growth and oocyte maturation in OrR-or CCAPR-depleted locusts are resulted from blocked uptake of Vg by developing oocyte s,but not by the declined synthesis of Vg in the fat body.Thus,it is of interest to further unveil whether OrR and CCAPR are involved in the regulation of patency by JH.When Mthl2,PTHR or TyR2 was knocked down,Vg protein levels were not significantly changed in the fat body,but decreased in the hemolymph and ovary,indicating that Mthl2?PTHR or TyR2 are unlikely to play key roles in patency functioning and Vg transportation to oocytes.In summary,an in vitro approach for live imaging of patency has been established in this dissertation research by using L.migratoria as a model system.Two GPCR genes,OrR and CCAPR were identified as candidates for the membrane signaling of JH and the regulation of patency by JH.These results provide the basis for further elucidating the molecular mechanisms of JH regulation in patency and consequent vitellogenesis and egg production in adult female insects.