Molecular Mechanism Of Chromatin Remodeling Complex PBAP Regulating The Onset Of Reproductive Diapause In Colaphellus Bowringi

Diapause is very common in animal kingdoms,allowing individuals to survive in adverse environmental conditions and maintain populations.In insects,diapause can occur specifically at the adult stage and is therefore termed reproductive diapause or adult diapause,characterized as reproduction arrest and large energy reserve storage.The lower level of juvenile hormone（JH）is the primary endocrine basis for reproductive diapause entry,and JH production in the corpus allatum is the sole source of JH.However,the upstream factors regulating JH production in reproductive diapause are still unclear.Therefore,clarifying the function of JH biosynthesis and investigating the upstream regulators of JH production are critical steps revealing the underlying mechanisms by which environmental conditions induce reproductive diapause.This study used the economically important horticultural insect pest,Colaphellus bowringi,as the model.At 25°C,16L:8D photoperiod can stimulate this cabbage beetle entering reproductive diapause.As a result,the diapause females exhibit ovary arrest and massive fat accumulation in their fat bodies.Conversely,12L:12D photoperiod induces reproduction,and the females develop with mature ovaries but store fewer lipids in the fat bodies.Given the clear diapause biology and significant diapause traits,I applied C.bowringi females in this work,determined the function of JH biosynthetic enzyme genes in diapause regulation and explored the upstream factors mediating JH production and reproductive diapause.The main results are as follows:1.Screening of the vital JH biosynthetic enzyme genes regulating reproductive diapause preparation in C.bowringiIn this study,we identified and cloned 22 JH biosynthetic enzyme genes in C.bowringi.At the nucleate and amino acid levels,these genes of C.bowringi are highly conserved compared with those of other insect species.Among of them,18 genes showed upregulation in the non-diapausing heads than diapausing heads.Knocdown of the 18genes highly expressed in the non-diapause females produced high RNAi efficiencies,but only RNAi of HMGR2,FPPS1,and JHAMT1 induced remarkable diapause traits,including ovary arrest and large fat accumulation.Moreover,knocking down HMGR2,FPPS1,and JHAMT1 decreased the expression levels of JH responsive genes,including Kr-h1,JHE1,and JHE2,and the expression changes of vitellogenesis gene Vgs and lipid-associated gene FAS2,FABP,and TGL1 are also consistent with the physiological status.Additionally,Methoprene,a JH receptor agonist,removed the suppressed expression of JH responsive genes induced by the RNAi（RNA interference）of HMGR2,FPPS1,and JHAMT1,and recovered the ovary development and lipid accumulation to the levels comparable with reproductive females.Hence,HMGR2,FPPS1,and JHAMT1are the vital JH biosynthetic enzyme genes regulating reproductive diapause preparation in C.bowringi.These results suggest that the transcriptional changes of HMGR2,FPPS1,and JHAMT1 are robust and dynamic molecular readouts of reproductive diapause,which provide the basis for the following study.2.The mechanism of PBAP complex regulating the onset of reproductive diapause in C.bowringiWe revealed 832 transcriptional regulators from C.bowringi transcriptome data（PRJNA338895）,among which 11 and 45 Unigenes were upregulated in the diapausing and non-diapausing females,respectively.By RNAi and Methoprene rescue experiments,Unigene5560 was identified as a candidate regulator of JH biosynthesis and reproductive diapause.Bioinformatic analysis showed that C.bowringi Unigene5560 is the orthologue of Drosophila melanogaster BAP170,a subunit of chromosome remodeling complex BAP/PBAP.BAP/PBAP complex contains eight core components,including BRM,BAP111,MOR,SNR1,BAP60,BAP170,Polybromo and Osa.BAP170 and Polybromo are the specific subunits of the PBAP complex,but Osa is the specific one of the BAP complex.RT-q PCR results suggested that most PBAP complex genes are highly expressed in the heads of non-diapausing female pupae and adults compared to diapausing individuals.Furtherly,the protein abundance of histone methylation H3K4me3 increased in the heads of non-diapausing female pupae and adults compared to the diapausing individuals.It has been demonstrated that H3K4me3 is a significant marker of active chromatin;therefore,it suggests higher chromosome remodeling activity and chromatin accessibility in non-diapausing female heads.Knocking down any of BRM,BAP111,MOR,SNR1,and BAP170 in non-diapause female pupae arrested ovary development and promoted fat storage.Expression of the reproductive diapause-associated genes was also changed to levels similar to diapause individuals.However,knockdown of Osa only inhibited ovary maturation but had no apparent effect on lipid accumulation.Thus,the BAP and PBAP complexes have distinctly different roles in the diapause regulation of C.bowringi.Based on the composition characteristics of BAP/PBAP complex,BRM was used to represent the common components of BAP and PBAP complexes,BAP170 to represent the PBAP complex,and Osa to represent the BAP complex for subsequent study.Knocking down any of BRM,BAP170,and Osa,in the non-diapause pupae decreased H3K4me3 levels in the heads of female adults,validating the regulatory activity of C.bowringi BAP/PBAP complex in chromosome remodeling.Meanwhile,Knockdown of BRM and BAP170 reduced the transcription of JH biosynthetic enzyme gene HMGR2 and JHAMT1,JHAMT1 protein levels,JH titers and the expression of JH responsive genes.However,RNAi of Osa had no significant effect on JH signaling.Further experiments showed that Methoprene removed the RNAi-induced diapause traits and gene expression changes.These results suggest that BAP and PBAP complexes can respond to photoperiods and regulate H3K4me3-asscociated chromosome remodeling,then mediate ovary development and lipid accumulation during diapause preparation.Particularly,the BAP170-mediated PBAP complex plays a decisive role in JH production and is a crucial regulator of reproductive diapause entry in C.bowringi females.3.The mechanism of PBAP complex regulating JH biosynthesis during the onset of reproductive diapause in C.bowringiAfter knocking down BAP170 in the non-diapause pupae,corpora cardiaca-corpora allata（CC-CA）complexes were dissected from the 24 hours-and 48 hours-old female adults and subjected to RNA-sequencing（PRJNA903668）.An overlap with 248 Unigenes downregulated by BAP10 RNAi was identified in both sampling time points.Based on the KEGG analysis,28 genes clustered into“Endocrine system”and“Metabolism of cofactors and vitamins”were considered the candidates which regulate the BAP170-mediated reproductive diapause.By using RNAi and Methoprene rescue approaches,CL2358.Contig1 was identified as a potential regulator linking PABP complex and reproductive diapause.Bioinformatic analysis showed that C.bowringi CL2358.Contig1 is the orthologue of calmodulin（Ca M）,which is highly conserved across insects.RT-q PCR results showed that Ca M was significantly more expressed in the heads of non-diapausing female pupae and adults compared to diapausing individuals.Knockdown of Ca M in non-diapause pupae considerably suppressed ovary development,increased lipid accumulation,and changed the expression of genes related to diapause and reproduction.Moreover,Ca M RNAi decreased JH signaling,indicated by the inhibition of JH biosynthetic gene expression and the downregulated levels of JHAMT1 protein,JH titers and JH responsive gene expression.When the females with Ca M RNAi were treated with methoprene,the decreased JH signaling and the diapause traits were reversed.The protein structure homology-modelling result revealed that C.bowringi Ca M possesses four Ca²⁺binding sites,suggesting its potential role in calcium signal transduction.We also found that Ca²⁺content in the head of 2d A non-diapausing female adults was significantly higher than that in diapausing individuals.After knockdown of BRM and BAP170,Ca²⁺content was significantly reduced in the head of 2d A female adults.Further analysis of the CC-CA transcriptome revealed that several calcium signaling genes,including Ca M,were downregulated by BAP170 RNAi.RT-q PCR validation of four selected down-regulated genes showed that ADCY3,Orai1,CACNA1A,and Ca M were significantly down-regulated in CC-CA tissue after BAP170 knockdown,as well as in the heads of female adults after knocking down BRM and BAP170.These results suggest that Ca M regulates the reproductive diapause of C.bowringi in a JH-dependent manner,and the BAP170-mediated PBAP complex can target Ca M expression to regulate JH production and eventually determine C.bowringi diapause entry.In summary,these results suggest that 1)transcriptional repression of JH biosynthetic enzyme gene HMGR2,FPPS1,and JHAMT1 is the endocrine basis for triggering reproductive diapause;2)chromatin remodeling complex BAP/PBAP regulates reproductive diapause preparation by mediating H3K4me3 modification and JH production;3)the BAP170-mediated PBAP complex controls Ca M expression in CA,which determines the onset of reproductive diapause.Therefore,“Photoperiod–PBAP complex–Ca M–JH”axis is the critical signaling cascade inducing reproductive diapause in C.bowringi.This work demonstrates the crucial role of JH biosynthetic genes in regulating insect reproductive diapause,establishes the key relationship of“Environmental cues–Epigenetic factors–Endocrine events”in the regulation of insect reproductive diapause.This study broadens our current perspective of diapause regulation,deepen the molecular ecological mechanisms of insect adaptation to the environments,and provide novel molecular targets and a theoretical basis for pest management.