Functional Analysis Of G Protein-Coupled Receptors In Vitellogenesis And Oocyte Maturation Of The Migratory Locust,Locusta Migratoria

Insects are well known with their high fecundity.Vitellogensis and oocyte maturation are prerequisite to egg production and embryonic development after oviposition.In many insects,the yolk protein precursor,vitellogenin(Vg)is synthesized in the fat body,transported into the ovary by hemolymph,and taken up by the developing oocytes.This process is controlled by juvenile hormone(JH),but the underlying mechanisms remain unclear.The migratory locust,Locusta migratoria has been served as an ideal model in studying the molecular basis of JH regulation in insect reproduction.Locust is a representative of hemimetabolous insects with panoistic ovaries,and its vitellogenesis and oocyte maturation are strictly controlled by JH.Moreover,the high fecundity is one of primary cues for locust plagues and outbreaks.G protein-coupled receptor(GPCR)is the largest membrane receptor family in eukaryotes,which is involved in regulating diversity of physiological processes.In addition,GPCRs are potential targets for insect pest control because of their variety of biological functions.Insect GPCRs are classified into four subfamilies: 1)Class A,rhodopsin-like receptors;2)Class B,secretin receptor;3)Class C,metabolic glutamate receptors;and 4)Class D,atypical receptors.It has been reported that GPCRs play pivotal roles in insect development,reproduction and behaviors.However,the function and regulatory mechanisms of GPCRs in insect reproduction have not been clearly revealed.Therefore,identification of key GPCRs and elucidation of their actions in insect vitellogenesis and oocyte maturation will help unveil the JH-mediated molecular machinery in insect reproduction.In this dissertation study,on the basis of genomic,transcriptomic and bioinformatic analyses,we selected 11 locust GPCR genes for further studies.These genes include a member of Class A: Neuromedin U receptor 1(NmUR1);9 members of Class B: G protein-coupled receptor 64(GPR64),G protein-coupled receptor 124(GPR124),Latrophilin 3(Lphn3),G protein-coupled receptor 143(GPR143),methuselah-like 14(Mthl14),methuselah-like 1(Mthl1),Methuselah 2(Mth2),Methuselah-like 5(Mthl5)and Methuselah 15-like(Mth15l),and a member of Class C: Orphan receptor-C(OrphanR-C).By using qRT-PCR,western blot and RNAi,we investigated the roles of above 11 GPCRs in Vg synthesis and transportation,ovarian growth and oocyte maturation.The observed phenotypes were divided into three categories: 1)inhibited Vg synthesis in the fat body;2)suppressed Vg secretion into the hemolymph;and 3)blocked Vg uptake in the oocytes.Under NmUR1 RNAi,the levels of Vg mRNA and protein were significantly reduced in the fat body,hemolymph and ovary.Consequently,the ovarian growth and oocyte maturation were suppressed.These observations illustrate that NmUR1 is likely to function in Vg synthesis.Depletion of Mthl1 caused significant decrease of Vg levels in all three tissues,accompanied by blocked ovarian development and oocyte growth.Interestingly,knockdown of Mthl14 led to reduced levels of Vg in the ovary and hemolymph but enhanced Vg accumulation in the fat body,suggesting a role of Mthl14 in Vg secretion from the fat body into hemolymph.Silencing of GPR124 or Lphn3 also led to decreased Vg levels as well as arrested ovarian development and oocyte growth.Strikingly,after GPR64 knockdown,Vg protein levels were remarkably increased in the fat body and hemolymph but significantly reduced in the ovary,while Vg mRNA levels remained similar.The ovarian development and oocyte maturation were also arrested in GPR64-depleted locusts.These data indicate that GPR64 plays a crucial role in Vg transportation into ovaries and uptake by oocytes.GPR143 RNAi resulted in significant decrease of Vg expression as well as blocked ovarian development and oocyte growth.OrphanR-C knockdown also showed defective phenotypes in Vg transcription,ovary development and oocyte growth inhibition.However,no defective phenotypes were observed with Mth2 RNAi.Unfortunately,the significant knockdown efficacy was not obtained with Mthl5 or Mth15 l,and further studies on Mthl5 or Mth15 l RNAi are needed.In summary,this study has identified 8 GPCRs potentially involved in the regulation of locust vitellogenesis and egg production,based on the phenotypes of Vg synthesis and transportation,ovarian development and oocyte maturation.The findings provide the basis for further studies of GPCR function in JH-dependent vitellogenesis and oocyte maturation,and help unveil the mechanisms of JH regulation in insect reproduction.