Study On Molecular Regulation Mechanism During Larval Molting Process In Silkworm

The insect cuticle is formed by the cross-linking of components such as chitin and cuticular proteins secreted by epidermis cells,which play a role in supporting the insect body,protecting the internal tissues and preventing water loss.However,the cuticle has only a limited capacity for expansion.To overcome the rigid constraints of exoskeletons,insects undergo periodic molting to accommodate growth in life span.Insect molting is driven by a series of sophisticatedly arranged events,including degradation of the old cuticle,formation of a new cuticle,ecdysis and pigmentation.Previous studies have shown that these molting processes are directly related to the titer of ecdysone in "pulse" manner.During the pre-molt period,the hormone titer in hemolymphe increases rapidly and then returns to normal levels after reaching a peak.Currently,studies demonstrated that 20 E response transcription factors is involved in regulation of molting process.However,the molecular regulatory mechanisms of molting process at the genome-wide level have not been investigated.The regulatory network in response to different hormone titer in the ecdysone pulse also needs to be identified,especially when the ecdysone titer decline,which hormone response factors are involved in the formation of new cuticle needs to be further investigated.In this study,we first analyzed the gene regulatory network at different developmental periods by temporal transcriptome from different larval molting periods of the silkworm.Further,key transcription factors in the pre-molt and post-molt periods were identified by ATAC-seq,and their functions in the larval molting process were verified.The main results obtained were as follows:1,Molting-related genes identified by time-series RNA-seqBy studying the histological changes of the epidermis during the fourth instar molt of the silkworm,we found that the degradation of the old cuticle occurred during the rise of the ecdysone titer(B and C1 stages);while the formation of the new cuticle occurred during the decline of the ecdysone titer(E1 and E2 stages).Based on temporal transcriptomic data,we identified genes associated with the old cuticle degradation and the new cuticle formation.During old cuticle degradation(B-D stage),proteases and chitin degradation related enzymes were significantly up-regulated.In contrast,cuticular proteins,chitin synthases,amino acid transport proteins and pigment synthesis genes involved in the new cuticle formation,were mostly highly expressed in E1 and E2 stages.Interestingly,cuticular proteins are arranged in clusters on the genome showed a typical co-expression phenomenon.In addition,if the ecdysteroid titers were artificially increased during E1 and E2,the expression of cuticle formation-related genes was significantly suppressed.Further,through gene co-expression network analysis,this study identified Bmftz-f1(Nuclear hormone receptor ftz-f1)and another novel transcription factor Bm CEBP(CCAAT/enhancer-binding protein)co-expressed with genes related to new cuticle synthesis,suggesting they are involved in formation of new cuticle.2,ATAC-seq identifies key regulators of new cuticle formationIn order to elucidate the regulatory mechanisms underlying the changes in transcript levels of molting-related genes,we conducted ATAC-seq to identify key regulators in the pre-molting(C1 stage)and post-molting(E2 stage)in the silkworm.We found that the genes adjacent to the open peak in C1 stage were mainly protein and chitin degradation genes,while the genes adjacent to the open peak in E2 were mainly cuticular protein genes.The most enriched motifs of peak in E2 corresponded to two transcription factors,Bmftz-f1 and Bm CEBP.Combined with the results of gene co-expression network analysis,genes involved in the new cuticle formation process were mainly regulated by Bmftz-f1 and Bm CEBP,and genes involved in the old cuticle degradation were mainly regulated by Bm ECR-USP and Bm GRH.3,Knockout of Bm CEBP and Bmftz-f1 causes molting defects in silkwormTo verify the effects of Bm CEBP and Bmftz-f1 on the formation of new cuticle in the silkworm,we performed functional studies on both by complete knockout(nosp-Cas9)and epidermis-specific knockout system(G25p-Cas9),respectively.Both knockout approaches confirmed that Bm CEBP and Bmftz-f1 are required for silkworm molting.Bmftz-f1 KO and Bm CEBPKO had serious molting defects,most of them died during molting process.Compared to control individuals,Bmftz-f1 KO and Bm CEBPKO＿G25p exhibited a reduced body size phenotype.We speculate that in knockout individuals,the expression of cuticular protein and chitin synthesis genes are down-regulated,resulting in molting defects and less extensibility of the newly formed cuticle.Bmftz-f1 KO and Bm CEBPKO＿G25p also exhibit difference in body color,possibly due to the expression change of pigment synthesis genes.We verified above hypotheses by comparing mutant with control individuals’ transcriptome using RNA-seq.By integrating multiple data,we found that Bm CEBP and Bmftz-f1 jointly regulated the expression of new cuticle synthesis related genes.By combining the above experimental results with multi-omics data such as RNA-seq and ATAC-seq,we carved out the molecular regulatory network of processes such as the old cuticle degradation and the new cuticle formation during the molting process of silkworm larvae,and identified two key factors regulating new cuticle formation,and finally confirmed their important functions in the molting process of larvae using knockout methods.Our results expand our knowledge of the molecular regulatory network of larval molt mediated by hormone pulse in insects,which has important fundamental theoretical implications.In addition,the study of the regulatory mechanism of larval molting will also help to explore new targets for pest control in agriculture and forestry,which has important practical applications.