| Mythimna separata is one of the important polyphagous agricultural pests which can break out and migrate.M.separata can migrate seasonally,and its annual migration from overwintering regions to non-wintering regions is the main cause of its sudden outbreaks and resultant disasters in recent years.In the 1960 s and 1970 s,China established that the overwintering boundary of M.separata was 0℃ isotherm(33°N)for January in the eastern part of China.However,due to climate changes,planting structure changes,China’s vast area,large temperature differences,the northern overwintering boundary is likely to change.Whether insects can overwinter in a region is determined by environmental temperatures and the physiological and biochemical substances in their bodies which are able to resist cold.These cold-resistant substances are related to the regulation of gene expression.In this research,the experimental sites are located in Hanzhong,Ankang and Yangling of Shaanxi Province,about 33 degrees north latitude.In 2015 and 2016,in each region the distances between samples were equal,and the laboratory-reared last-instar larvae of M.separata were put in population cages in fixed locations in November.The survival rate of M.separata was investigated every month until March of the following year.During monthly sampling,changes in the contents of water,glycogen,trehalose and triglycerides in M.separata were examined by biochemical techniques.Hsp70,Hsp90,AKH1 and AKH2 genes,which are associated with cold resistance,were cloned by molecular biology techniques,and the relative expression of the above four genes of overwintering M.separata in different periods was quantitatively analyzed by real-time PCR.The northern overwintering boundary of M.separata in Shaanxi Province was identified for the first time,and the changes in M.separata cold-resistant substances in winter and the mechanisms of gene regulation and expression were clarified.The results are summarized as follows:1.Two years of experiments on the survival rates of overwintering M.separata showed that M.separata in southern Shaanxi(33° north latitude)can overwinter,but its survival rate was low.From winter 2015 to spring 2016,the highest survival rates of armyworms were 3.77%,4% and 0% in the Hanzhong area,Ankang area and in Yangling,respectively.From winter 2016 to spring 2017,the highest survival rates of armyworms were 2.33%,1.83%,and 0.5% in the Hanzhong area,Ankang area and in Yangling,respectively.The survival rate of overwintering armyworms showed a decreasing trend,and adult M.separata can be found in Hanzhong and Ankang areas at the end of each winter.2.The changes in the contents of water,glycogen,trehalose and triglycerides in overwintering M.separata were tested monthly.We confirmed that glycogen,trehalose and triglycerides were quickly consumed during the process of overwintering.The amount of glycogen decreased from 52 mg/g to 40 mg/g from November to January,and the trehalose content decreased from 35 mg/g to 30 mg/g.Triglycerides were also consumed as energy substances.During winter 2015 from November to January the triglyceride content decreased from 38 mg/g to 34 mg/g.However,from January to March the triglyceride content decreased from 34 mg/g to 20 mg/g.In winter 2016 from November to January the triglyceride content decreased from 37 mg/g to 32 mg/g,and from January to March the triglyceride content dropped from 32 mg/g to 18 mg/g.Water content in the early winter dropped rapidly.During winter 2015 from November to January the water content decreased from 78% to 68%,and it decreased from 68% to 62% from January to March.In winter 2016 from November to January the water content decreased from 77% to 68%,and it decreased from 68% to 62% from January to March.3.The conserved region sequences of Hsp70,Hsp90,AKH1 and AKH2 genes were obtained by PCR cloning: Hsp70 sequence 1962 bp,Hsp90 sequence 2154 bp,AKH1 sequence 343 bp,AKH2 sequence 479 bp.4.Expression of the four genes above during overwintering were detected by real-time PCR.The results showed that Hsp70,Hsp90,AKH1 and AKH2 can express in the overwintering process,but the expression levels were different.The expression of the Hsp70 gene was lower than that of the Hsp90 gene.Differences in Hsp70 and Hsp90 gene expression in the two cities may be attributed to minor differences in climate.During the overwintering period,although the expression of the AKH1 gene was low in the early stage,it increased gradually.The expression level of the AKH2 gene was high in December,decreased in January,and rose in February and March.5.The results showed that during the overwintering period the expression of Hsp70 and Hsp90 increased,and the content of glycolipid decreased,which indicates that the mode between Hsp70,Hsp90,and glycolipid metabolism is negatively regulated.Changes in the expression of AKH1 and AKH2 in vivo indicate that M.separata can resist low temperatures by regulating its glycolipid metabolism.The large expression of AKH2 in the early stage of M.separata larvae led to a sharp decline in glycogen in the early stage of overwintering.And the large expression of AKH1 in the late overwintering process made M.separata use triglycerides as an energy substance,which indicates that the mode(same comment)between AKH1,AKH2 and glycolipid metabolism is positively regulated.This research shows that M.separata can overwinter successfully at 33°N in Shaanxi Province and clarifies the changes in expression of the Hsp70,Hsp90,AKH1 and AKH2 genes and cold-resistant substances in overwintering M.separata.This study lays a foundation for further research on the molecular mechanism of substances related to cold resistance of M.separata and reveals the ecological adaptability of M.separata at the molecular level. |
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