| Climate change often leads to the fluctuation of insect populations in frigid and temperate zones.Low temperature in winter is the direct obstacle for survived insects.Their cold-tolerance capacity is one of the key factors that restrict occurrence,distribution and diffusion of insect populations.The Chinese white pine beetle,Dendroctonus armandi Tsai and Li,is arguably the most destructive forest insect in the Qinling Mountains in Northern China.Therefore,it’s important to research the low temperature tolerance of D.armandi for early warning about potential distribution and its integrated control.Thus,we focused on the overwintering Chinese white pine larvae physiological indices,relative gene expressions and cold-tolerance.This research included these aspects: testing overwintering physiological indices and enzyme activity changes;researching relative cold-tolerance genes’ expression profiles in different temperatures with different treating time;examining the responses of dsRNA-injected,water-injected,and non-injected D.armandi larvae to low temperature by mortality analysis;investigating the composition and the roles of the microbial communities(bacteria and fungi)present in the guts of D.armandi larvae in overwintering stage.The mainly results were listed as follows:1.Analysis of supercooling point(SCP)changes of overwintering D.armandi larvae,as well as mortality at low temperatures to assess cold tolerance.The SCP tended to decrease initially as the ambient temperature decreased until the middle of the overwintering period before increasing until the end of the winter as the environmental temperature increased.The minimum monthly mean appeared in January.Gradually decreasing temperatures in autumn resulted in a continuing reduction of the SCP and an enhancement of cold hardiness that was demonstrated by increased low temperature survival.The ability of D.armandi larvae to survive at low temperatures improved constantly as the temperature decreased in the environment.The cold-tolerance ability enhanced after the cold acclimation treatments of D.armandi larvae.2.The values of physiological indices and the enzyme activities involved in the overwintering stage were studied in D.armandi larvae in each month during the winter.The determination of physiological indices and enzyme activities showed: 1)Trehalose,sorbitol and glycerol contents were considered as the potential cryoprotectants,which showed significant negative correlations with the SCP;2)Glycogen content was considered as the important energy substances,which decreased obviously in mid-overwintering period;3)The total water content of larva body changed little during the whole overwintering period.At the same time,the bound water and free water transformed into each other,which implied that "Water-saving mechanism" might have been conducted to improve the cold tolerance of D.armandi larvae;4)The activities of four enzymes(SOD,CAT,LDH and AchE)decreased,whereas POD,PK and MDH showed the opposite trends.Other enzyme activities(TPS,SDH and GLK)were low during the overwintering period and later increased and stabilized during spring.3.Real-time PCR analysis was used to detect the transcript levels of DarmTPS,DarmSDH and DarmGLK in overwintering D.armandi larvae among different low temperatures with different treating time,and the DarmTPS,DarmSDH and DarmGLK genes were knocked down by RNAi to study the influence on mortality.The results showed the levels of DarmTPS and DarmSDH were up-regulated,by contrast,the DarmGLK expression level decreased notably during the overwintering stage.The expression levels of these three genes were higher in December than that in May.The expression level of dsRNAi-TPS,dsRNAi-SDH and dsRNAi-GLK dropped significantly,while,the transcript levels of DarmTPS,DarmSDH and DarmGLK in the non-injected and water-injected overwintering D.armandi larvae remained unchanged.After low-temperature exposure,the mortality of the dsRNA-treated larvae was remarkably higher than that of the water-injected and non-injected controls.4.Real-time PCR analysis was used to detect the transcript levels of heat shock proteins(DaHSP90,DaHSP70 and DasHSP)and cold shock protein in overwintering D.armandi larvae among different high and low temperatures with different treating time.The results showed levels of DaHSP90 highly expressed under low temperature treatments,DaHSP70 highly expressed under high temperature treatments.Furthermore,significant up-regulation of DaHSP90 and DasHSP was observed with long-term temperature acclimation,DaHSP70 showed higher expression level with short-term temperature acclimation.The expression level of DarmCSP(D.armandi cold shock domain-containing protein)was up-regulated during the overwintering stage and DarmCSP could be induced by low temperature.The expression level of oversummering adults was also up-regulated when beetles were exposed to low temperatures.However,the expression level was lower than that of overwintering larvae.5.The Illumina MiSeq sequencing method was employed to investigate the composition,diversity,dynamics variation regularity and the roles of the microbial communities present in the guts of D.armandi larvae at different month during overwintering period.The microbial community structure was simple of overwintering D.armandi larvae.The intestinal microbial community structure changed with the change of environmental temperature.As ambient temperature declined,Proteobacteria(mostly γ-proteobacteria)became the predominant phylum in the larval gut,while followed with Actinobacteria,Firmicutes and Bacteroidetes,respectively.The results suggested that the gut bacterial community compositions increased in abundance in November and December compared with that in October and January.Zygomycota,Basidiomycete and Ascomycete constituted fungal phylum in the larval gut.Ascomycete was the dominated fungal phylum in overwintering D.armandi larvae gut.These results suggested that the microbial communities might have protective effects of overwintering D.armandi larvae to resist low temperature stress. |
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