The Functions Of MAPK Signaling Pathway In The Reproduction In Female Colaphellus Bowringi

Reproduction is vital for population maintenance in insects.The regulatory mechanism in reproduction of female insects has always been hot issue in the field of entomology.Previous studies showed that the mechanism of reproductive regulation varies greatly among different insects,and at present,there are only systematic studies in model insects.MAPK signaling pathway is an evolutionarily well-conserved intracellular signal transduction pathway,which plays an important role in mammalian reproductive development.However,its role in female insect reproduction is poorly understood.Therefore,this study explored the function and molecular mechanism of MAPK signaling pathway in female insect reproduction,implemented on Colaphellus bowringi,an important pest on cruciferous vegetables.The main results are as follows:1.Cloning and sequence analysis of candidate genes in MAPK signaling pathway of C.bowringiIn this study,17 candidate genes were cloned and identified,came from extracellular regulated protein kinase(ERK),c-Jun N-terminal kinase(JNK)and p38 MAPK(P38)pathways,which are the most important pathways.Phylogenetic analysis showed that candidate genes were most closely related to Coleopteran corresponding genes.Compared to the corresponding gene domains of other species,it was found that the functional domains were the same and conserved.2.The RNAi screen of key genes in MAPK signaling pathway of C.bowringiCandidate genes silencing was performed through injecting dsRNA into 3-day female pupae.According to ovarian development grades,we found that silencing MAPK-ERK kinase(MEK),ERK,kinase suppressor of RAS 2(KSR2),profiling(PFN),and P38 inhibited ovarian development.Thus,these 5 genes were termed as key gnes of female reproductive regulation in C.bowringi,for following studies.3.The spatiotemporal expression of key genes in MAPK signaling pathway of C.bowringiThe expression levels of key genes in the heads,fat bodies,midguts and ovaries of female C.bowringi at 4-day post-ecolosion(PE)were detected.The results indicated that key genes were highly expressed in the ovaries.The expression patterns of key genes were different in the ovaries at 0,2,4 days PE.MEK and P38 expressed highest at 0-day PE.The transcript levels of ERK and PFN were basically unchanged,while the expression of KSR2 was highest at 4 days PE.These results implied that key genes play an important role in ovarian development,but the function time is different.4.Effects of key genes in MAPK signaling pathway in female survival and reproduction of C.bowringi(1)The effects of key genes on the ovarian development of C.bowringi were determined by RNAi and q RT-PCR,including the analysis of ovarian development grade,the determination of ovarian size and the expression of genes related to vitellogenin synthesis.The results showed that the 5 key genes are very important for ovarian development,which may regulate ovarian development by affecting vitellogenesis;(2)After silencing key genes,the results of survival rates and egg production showed that the key genes significantly reduced the longevity and fecundity of female adults,which revealed the importance of key genes in survival and fecundity;(3)U0126,a commercialized specific inhibitor of ERK pathway,was injected to inhibit ERK phosphorylation.The results showed that U0126 treatment had no effect on the survival and ovarian development of C.bowringi,suggesting that U0126 might lack activity in C.bowringi.5.Effects of key genes in MAPK signaling pathway on hormonal signaling,cell cycle and germline stem cell of female C.bowringiIn this study,three key genes,ERK,PFN and P38 were used to represent ERK,JNK and P38 pathways respectively,and the molecular mechanism of their regulation of female reproduction in C.bowringi was explored.(1)Silencing ERK and PFN significantly down-regulated the transcription levels of 20-hydroxyecdysone(20E)biosynthetic genes,but had no effect on the expression of juvenile hormone synthase.Further,after cutting off juvenile hormone signal,it was found that only ERK expression was significantly decreased,suggesting that juvenile hormone might regulate female reproduction of C.bowringi through ERK pathway.(2)Only RNAi of ERK down regulated the expression of cyclin D(CCND),a key regulator of Gap 1 phase(G1)in the cell cycle.Only after silencing PFN,the expression levels of DNA replication related genes reduced and the nuclear size also decreased,indicating that ERK pathway affected G1 and JNK pathway affected the synthesis phase(S).(3)Knockdown of ERK,PFN and P38 reduced the expression of germline stem cell(GSC)marker genes,suggesting that they played potential roles in GSC development and differentiation.In conclusion,after RNAi screening,this study indicated that MEK,ERK,KSR2,PFN and P38 significantly affect the survival,ovarian development and oviposition of C.bowringi.After further exploring the regulation mechanism,it was found that ERK and JNK pathways may be involved in 20 E biosynthesis and cell cycle regulation,while P38 pathway mainly affected the development and differentiation of GSC.Our work provides new clues for further understanding the reproductive regulation mechanism of female insects and new molecular targets for pest control.