The Functions Of Circadian Clock Genes In The Reproductive Diapause Regulation In Colaphellus Bowringi

Diapause is a state of arrested development induced by photoperiod or temperature that allows insects to survive in adverse environmental condition.Perceiving the diapause-inducing stimuli in advance and carrying out appropriate behavioural activities or physiological processes is essential for insect to successfully survive the adverse seasons.Understanding how insect initiates facultative diapause and prepares diapause can provide us new insights to study the molecular mechanism of seasonal adaptation strategy.It has been shown that the circadian clock genes can participate in photoperiodic measurement and regulate reproductive diapause initiation through JH signaling in short-day-induced winter diapause.However,how circadian clock genes translate photoperiodic information into downstream hormone signaling for diapause destiny and then affect diapause preparation remains largely unknown.The cabbage beetle,Colaphellus bowringi Baly,a serious pest of cruciferous crops,is widely distributed in of China,such as Jiangxi,Shandong and Heilongjiang.It can be reliably induced to undergo facultative reproductive diapause by exposure to long-day condition or low-temperature.The cabbage beetle is sensitive to the photoperiodic stimuli during the larvae stage,and individuals pre-program their developmental destiny according to the photoperiodic signals that they received.Since the adults emerged out,the beetles undergo diapause preparation phase according to developmental destiny that formed in the diapause induction stage.In this study,the C.bowringi high-diapause(HD)strain collected in Xiushui,Jiangxi Province(29°10'N,114°40'E)were used in our study.And experiments,such as real-time PCR,RNAi and RNA-Seq,were used to investigate the functions of circadian clock genes in photoeriod regulating reproductive diapause.The main conclusions are as follows:1.Screening the candidate circadian clock genes that involved in the diapause regulation of C.bowringi.The non-photoperiodic-diapause(NPD)strain of C.bowringi that developed in the laboratory by artificial selection,could not enter reproductive diapause under long-day photoperiod.Hence,comparing the 24-hour expression patterns of circadian clock genes between HD and NPD strains would contribute to screen candidate circadian clock genes that involved in diapause regulation of C.bowringi.First,we successfully cloned 8 circadian clock genes(clock,cycle,period,timeless,cryptochrome2,double-time,shaggy and vrille)and obtained intact ORFs of 6: clock,timeless,cryptochrome2,double-time,shaggy and vrille.The domain inference and multiple domain sequence alignments suggested that the domain types and sequneces of C.bowringi circadian clock genes were consistent with those of the corresponding gene in other species.Blastp alignment and phylogenetic trees analyses indicated that the circadian clock genes of C.bowringi show remarkable homology with the circadian clock genes in Tribolium castaneum and Leptinotarsa decemlineata.The quantitative real-time PCR indicated that clock,cycle,period,timeless,cryptochrome2 and vrille were differentially expressed between HD and NPD strains reared under long-day condition during the diapause induction phase.These findings suggested the potential relationship between circadian clock genes and long-day-regulated summer diapause regulation in C.bowringi.2.The regulation mechanism of candidate clock gene in diapause induction of C.bowringi.The non-24-h light-dark cycles experiments show the duration of scotophase is essential for the determination of C.bowringi diapause.In this study,we chose period,a circadian clock negative regualtor that highly expressed during the scotophase in C.bowringi,and its complexes member timeless and cryptochrome2 for furture study.The results showed period,timeless and cryptochrome2 played important roles under diapause inducing long-day condition.We respectively knocked down period,timeless and cryptochrome2 in the 3-day-old larvae(most sensitive to photoperiod),and dsgfp treatment was served as a control.Under the diapause-inducing photoperiod(16L : 8D),knocking down period and timeless significantly decreased the rate of burrowing behavior while knocking down cryptochrome2 only decreased the lipid accumulation during the diapause preparation phase.More than 50% female beetles of the period and timeless RNAi showed developed ovary and decreased lipid accumulation.The JH biosynthesis genes(HMGR2,FPPS1 and JHAMT1)and JH-induced genes(Kr-h1,JHE1,Vg1,Vg2 and TGL1)significantly increased in the females with suppression of period and timeless during diapause induction phase.The lipogenesis genes(ADH,ALDH1,FAS1 and TKT2)and stress resistance genes(DP1,HSP21,HSP23,HSP70 and SOD)that suppressed by JH were significantly downregulated.It implied that suppression of period and timelss during diapause induction phase could activate the JH biosynthesis signaling in diapause preparation phase and then regulate the lipid accumulation,ovary development and stress resistance in the female adults.To explore how period and timeless translated photoperiodic information into downstream JH signaling,we used RNA-Seq to analyize gene expression profiles after period and timeless RNAi.After RNA-Seq,differential expresses genes(DEGs)analysing,time series and WGCNA analysing were performed,the results showed period and timeless likely transformed the photoperiodic signals that received during diapause induction into downstream JH synthesized signals through DEGs,such as larval serum protein 1 ? chain?glutenin?trypsin ??ctp synthase?histone H3/H4-like?pyruvate kinase.3.The regulation mechanism of candidate circadian clock gene in diapause preparation of C.bowringi.In insects,photoperiodic signals received during the diapause induction phase have irreversible effect on diapause initiation.Insects continue to be exposed to diapause-inducing photoperiod in the diapause preparation phase(DPP)for diapause preparation.However,how photoperidic signals experienced during the DPP regulate diapause remains largely unknown.In this study,we found that the lipid content of C.bowringi female decreased after diapause-destined(DD)individuals were exposed to a diapause-inhibiting photoperiod(12L : 12D)during the DPP.The circadian clock negtive regulators period and timeless were probably involved in the photoperiodic response of beetles during the DPP.Period and timeless presented obvious oscillation of circadian rhythm and photoperiodic response during the DPP in DD females and knock-down of these genes in DD females caused their lipid content to decrease.Period and timeless probably promote lipid accumulation by regulating the expression of genes that regulate lipogenesis and lipolysis.Moreover,decreased lipid accumulation caused by exposure to different photoperiods during the DPP was independent of juvenile hormone.The lipid content also decreased in individuals injected with ds Met after the beetles were trasnsformed to short-day condition during DPP.In summary,these results suggest that photoperiodic signals received during the DPP influence lipid accumulation in DD insects.DD insects still have some ability to monitor photoperiodic changes during the DPP and period and timeless are probably involved in regulating physiological responses to photoperiodic signals during diapause preparation.In this study,we cloned and screened the circadian clock genes that probably involved in C.bowringi diapause regulation.And we reported period and timeless involved in regulating the diapause induction and diapause preparation in long-day induced summer diapause insect for the first time.In the diapause induction phase,period and timeless could transform the photoperiodic signal into downstream JH synthetise signal through genes including larval serum protein 1 ? chain?glutenin?trypsin ??ctp synthase?histone H3/H4-like?pyruvate kinase.In the DPP,DD beetles could use the photoperiodic signal to regulate lipid accumulation.Period and timeless transformed the photoperiodic signal to the expression of genes that regulate lipogenesis and lipolysis and then regulated the lipid accumulation.These results provide new evidences to circadian clock genes involved in diapause regulation of long-day induced summer diapause insect.It not only provide new clues for revealing the molecular mechanisms of how photoperiodic signals regulating insect diapause,but also is helpful to further understand the mechanisms of insect seasonal adaptation and evolution.