Respiratory&Survival Dynamics And Differential Expression Of Trehalases In Diapausing Pupae Of Pieris Melete

The cabbage butterfly,Pieris melete Ménétriés?Lepidoptera:Pierdidae?is a main defoliator pest of cruciferae vegetables,such as cabbage and radish.The studied of this thesis based on the prophase studies about cabbage butterfly on environmental control,photoperiod control,photoperiod measurement mechanism,carbohydrates metabolism,during winter and summer and differential expression of heat shock protein,etc.in the lab,a method was established that can detect respiration simply and quickly,monitoring the respiratory dynamics of diapause pupae in summer and winter under different temperature,and observe the survival dynamic under the different temperature at the same time,than select glucose metabolism?trehalase?genes associated with diapause,cloning two complete sequences of trehalase,analysing differential expression of mRNA under different temperature and in different diapause stage,in order to further understand the respiratory and energy metabolism in response to adverse conditions in the process of insect diapause,it provides a reference for exploring the ecological adaptation mechanism of pests.The results are as follows.Based on the Sable small animal respiration measurement system,two tests ways of the respiration chamber with the 8-channel gas circuit converter configured by Sable respiration measurement system and using the Luer adapter syringe as replaced respiration chamber to monitor the insects respiratory metabolism.The results showed that the O₂consumption in Helicoverpa armigerapupa was 0.2425?±0.0143?mL/g·h and 0.2389?±0.0146?mL/g·h,respectively.The CO₂ release volume was 0.1562?±0.0098?mL/g·h,and 0.1639?±0.0092?mL/g·h,respectively.The application of Luer adapter syringe as alternative-breathing chamber,compared to self-contained breathing chamber of Sable measurement system,application of alternative respiration chambers significant saving the test time?2.75 h for 21 samples?compared to 2.30 h for 7 samples per test cycle,when using the 8-channel gas circuit and its respirator.Using alternative Luer adapter syringe to monitoring the respiration,based on the CO₂ release dynamics,it is easy to distinguish differences in respiratory metabolism for different instars or developmental states.The respiratory metabolism during diapause in summer and winter was measured by Sable small animal respiration measurement instrument,It shows that the respiratory metabolism rate presented a u-shaped curve under the appropriate temperature conditions,and the trend was relatively insignificant under the high temperature and low temperature conditions.during the whole diapause period,the respiration rate is relatively high in pre-diapause?1d and 5d?,it was lowest during the diapause,some individual,the rate of some pupa rise in post diapause.Respiration rate was significantly different in different diapause stages at different temperatures,In the initial stage of diapause,the respiratory metabolic rate increases significantly with the increase of treatment temperature?P<0.001?.the respiration rate during the diapause keep in a low level at 5??V<0.05 ul/mg/h?,and the rate get lower when the diapause goes further,it keep in a relatively low level during the whole diapause stage at 30?.During the diapause of winter and summer diapausing pupa of Pieris melete,there are individuals dead gradually,and the survival rate tends to decrease with the increase of temperature,A large number of summer diapausing pupa died rapidly at high temperature,The final survival rate was extremely low after 120 days afterpupate,18.8%at 25?and0.0%at 30?;the rate of diapausing pupa in winter was higher than in summer,it's lowest120 days after pupate at 20??72.5%?,The difference of survival rate between different temperatures was smaller than that of summer diapause.Log-rank?Mantel-Cox?test was used to analyze of survival of pupae in different periods,the result shows:The effect of temperature on survival rate of Pieris melete Ménétriés in summer and winter was very significant.In the study,two homology full length genes of trehalase were cloned from P.melete.The sequence analysis showed that these two genes contained the conserved domain of trehalase.There were two transmembrane domains in trehalase B,while there was no in trehalase A.And it is showed that there was evolutionary divergence between trehalase A and trehalase B based on the phylogenetic tree analysis.The result of qPCR indicated that the expression levels of trehalase A and trehalase B were low in the pre-diapause period,and then increased in the post-diapause period.