Effects Of Parasitism By Cotesia Vestalis On Trehalose Metabolism Of Plutella Xylostella And Preliminarv Exploration Of Molecular Mechanisms

Trehalose is the major sugar in the hemolymph of many insects and it plays a cruial role in regulation of insect reproduction,growth,and energy storage and utilization.When P.xylostella are parasitized by C.vestalis,the hosts show inmmune defects and a metabolic disorder,as well as the changes in hemolymph sugar levels.In this thesis,firstly,the concentration of trehalose and glucose were detected in both parasitized and non-parasitized P.xylostella larvae.Next,the genes of six enzymes which are related to the insect trehalose metabolism were cloned,and qPCR and RNAi methods were used to verify that the expression of genes encoding some enzymes of trehalsoe biosynthesis were upregulated in P,xylostella after C.vestalis parasitization.Moreover,trehalase(a trehalose degradative enzyme)activity showed no significant changes between non-parasitized and parasitized larvae.Finally,pseudoparasitization and microinjection experiments suggested that the venom played important roles in increasing the trehlaose concentration in early instars of parasitized hosts,besides,the venom could also elevate the transcriptional level of PxHK after parastization.The results are summarized as follows:1.The middle 3rd instar(3rd-?)were setted as an initial time-point,the concentration of the P.xylostella hemolymph trehalose and glucose were detected in late 3rd(3rd-?)instar,early 4th(4th-I)instar,middle 4th(4th-?)instar and late 4th(4th-?)instar both in parasitized and non-parasitized C.vestalis larvae.The results showed that the trehalose concentration in parasitized larvae was higher than the one in non-parasitized larvae in all the above instars,repectively.However,the glucose concentration of parasitized larvae was only higher than that in non-parasitized larvae in rd-? and 4tth-? instar,respectively.2.Based on the data of P.xylostella genom and transcriptome,the genes of six enzymes which are related to trehalose metabolism were successfully cloned.There were four trehaose synthase-related genes(PxGP,PxHK,PxTPS),one trehalose transporter gene PxTRETl and one trehalose degradative gene PxTRE-2.The qPCR was used to detect the effect of parasitism on these genes.The results suggested that the transcriptional level of PxHK was higher in parasitized larvae than that in non-parasitized larvae in 3rd-III instar;the transcriptional level of PxTRET1 was higher in parasitized larvae comparing with non-parasitized larvae in 4th-? instar,meanwhile,the transcriptional level of PxGP was lower in parasitized larvae than that in non-parasitized larvae;the transcriptional levels of PxGP,PxTPS,PxHK were higher in parasitized larvae comparing with non-parasitized larvae in 4th-? and 4th-?larvae;however,the transcriptional level of PxTRETl was lower in parasitized larvae than that in non-parasitized larvae.The transcriptional level of PxTRE-2 showed no significant difference in all these instars.Furthermore,when analyzing the trehalase activity in parasitized and non-parasitized larvae,we found that the parasitism had no effect on the trehalase activity.3.PxTPS was choosed as a target gene to detect its molecular function in affecting the trehalose concentration in parasitized P.xylostella larvae.Parasitized larvae in 3rd-? instar were microinjected with 500ng dsPxTPS,then the transcriptional level of PxTPS and the concentration of hemolymph trehalose were detected in 4th-? P.xylostella instar.The results showed that the transcriptional level of PxTPS was significantly lower in dsPxTPS microinjection than in dsGFP microinjection,which indicated quite good RNAi knockdown efficiency.The trehalose concentration of dsPxTPS microinjected larvae was lower than that of the dsGFP microinjected one,which implied that the much higher transcriptional level of PxTPS induced by parasititsm was the reason of higher trehalose concentration in hosts.4.In order to dissect the influence of parasitic factors on the host's hemolymph trehalose concentration,pseudoparasitism,CvBV and venom microinjection were used to confirmed it.The results suggested that the trehalose concentration was much higher in pseudoparasitized larvae than in non-parasitized larvae in 3rd-? and 4th-?larvae,but showed no obvious difference in the later instars;the results of microinjection of CvBV and venom showed that venom played a crucial roles in maintaining a higher trehalose concentration in 3 rd-? instar,while CvBV had no effect.The qPCR suggested that the higher trehalose concentration in 3rd-? P.xylostella larvae was because of the increasing expression level of PxHK after the C.vestalis veom challenging.