The Effect Of Circadian Clock Genes On The Rhythm And Reproductive Diapause Of Harmonia Axyridis

Ladybird Harmonia axyridis(Coleoptera;Coccinellidae)is a completely metamorphosis insect species;and due to its strong environmental adaptation,high fecundity,long life-span,euryphagy and hearty appetite,it is normally used as predatory natural enemy for biological control.H.axyridis will enter diapause in sensing of the temperature changes in early winter,for safely overwintering in the field.Therefore,diapause could be artificially induced by modifying thermal conditions,low temperature and short photoperiod treatments,in rearing of H.axyridis.Artificial regulation of diapause in H.axyridis has important application value,but the mechanism of signal engendering and transmission in diapause is not clear.Photoperiod and temperature changes are key external factors that stimulate diapause for H.axyridis.The rhythm behavior mainly depends on the circadian clock to respond to the external environment,especially the photoperiod changes.The circadian clock participates in and drives a series of rhythm feedback processes from gene expression to individual behavior by means of regulating downstream physiological systems directly.Diapause of H.axyridis is also regulated by circadian clock.How the external photoperiod signal is converted into hormone signal,which leads to the development change of relevant parts,and finally shows the change in the level of diapause behavior,this problems have not been reported yet.We have carried out research and experiments on the above problems.First of all,by dissecting the reproductive system of the female adult of H.axyridis and quantifying Vg,the specific differences of female H.axyridis in long photoperiod and short photoperiod at 20? were studied.Then,a complete day and night sampling of female adult H.axyridis reared under long photoperiod and short photoperiod conditions is carried out through transcriptome sequencing technology,thus obtaining the expression of daily rhythm of five circadian clock genes under long photoperiod and short photoperiod conditions;Finally,through the screening of differentially expressed genes between transcriptome samples with different photoperiods,genes that may play a role in the conversion process from circadian clock signal to hormone signal were found and verified by quantitative PCR.That laying a good foundation for the later gene function verification.Through the above research,we have obtained the following research results:1.Under the condition of 20?,anatomy shows that female H.axyridis in long photoperiod showed ovarian developmental signals on the 6th day after eclosion,mature eggs were found in ovarian tubules on the 9th day,while female H.axyridis in short photoperiod showed ovarian developmental signal on the 15 th day after eclosion.Through quantitative PCR of Vg,it was found that the Vg expression of female adults of H.axyridis increased day by day under long photoperiod conditions,that is,the accumulation of vitellogenin increased day by day,while the accumulation of vitellogenin under short photoperiod conditions did not increase day by day as the same with under long photoperiod conditions,but showed a trend of increasing first,then decreasing and then increasing.2.Through transcriptome sequencing,it was found that among the five major circadian clock genes,the expression of period and timeless were higher at night,while the expression of cycle,clock and cryptochrome 2 were higher during the day.3.Through the screening of differentially expressed genes between transcriptome samples of different photoperiods,we found VMAT(vesicular monoamine transporter)used to transport dopamine between different cells in the process of nerve signal transmission,and found that it may play a role in the process of circadian clock signal to hormone signal conversion.To sum up,we have obtained the specific differences of female H.axyridis in long photoperiod and short photoperiod at 20?.The daily rhythm of expression of five biological clock genes under long and short photoperiods was also obtained.The VMAT gene that may play a role in the conversion from circadian clock signal to hormone signal was screened out.These will lay a good foundation for promoting the clarity of diapause mechanism of H.axyridis,and also provide theoretical reference for the breeding and application of H.axyridis.