The Research Of Insecticidal Mechanism Of Destruxin A Against Bemisia Tabaci

Bemisia tabaci(Gennadius)(Hemiptera:Aleyrodidae)is a serious pest of agricultural crops in different regions of the world.The injudicious use of chemical pesticides has resulted in the development of insecticide resistance by B.tabaci.Developing bio-pesticide is very urgent for the control of B.tabaci.Destruxins are secondary metabolites produced by entomopathogenic fungi.Due to the high insecticidal activity,destruxins have great application potential in biological control of B.tabaci.However,the insecticidal mechanism of destruxins is still not clear.In this study,the Transmission Electron Microscope(TEM)technique was used to obtain the pathological characteristics,and transcriptome sequencing was used to construct the differentially expressed genes database of B.tabaci after destruxin A treatment.The activity of detoxification enzymes and content of energy substances of B.tabaci after destruxin A treatment were also measured.The main results are as follows:1.Adults of B.tabaci were feed by LC₅₀ concentration of destruxin A.After treated by4 hours,8 hours and 12 hours,the ultrastructure of fat body and midgut was analyzed by transmission electron microscopy.The results showed that the midgut cells of the B.tabaci adults were severely damaged after destruxin A treatment.The vacuolization of the cells accrued and the nuclear membrane were swelling in the early treatment stage.At the later stage of treatment,all the microvilli fell off and the contents of the organelles died out.The fat body cells of B.tabaci adults became lighter color in the early stage of destruxin A treatment and the lipid droplet membrane structure became transparent.Moreover,a large number of vacuoles appeared.At the later stage of treatment,the lipid droplet membrane structure collapsed.2.Adult B.tabaci were treated by LC₅₀ concentration of destruxin A by 4 h,8 h,and 12h,and then the transcriptomes of destruxin A treated and control of B.tabaci adults were sequenced by Illumina 4000 platform.The results showed that multiple differentially expressed genes were identified between the treated and the control group.There were 637 genes up-regulated and 1071 genes down-regulated at 4 hours after treatment;336 genes were up-regulated and 280 genes were down-regulated at 8 hours after treatment;334 genes were up-regulated and 221 genes were down-regulated at 12 hours after treatment.Functional annotation of differential genes revealed that most of the genes involved in transport and metabolism,signal transduction,cancer-related,detoxification of foreign substances,immune system and other processes,indicating that destruxin A could affect multiple pathways in B.tabaci.Further analysis indicated that these differential genes were mainly concentrated in pathogen infection and immune system,cells Process and metabolism and other related pathways,such as the chemokine signaling pathway,apoptosis,carbon metabolism.3.By comparing the destruxin A treated and the control transcriptomes,a total of 40 differentially expressed genes of detoxification were detected,including 29 CYP450 genes,5 GST genes,and 6 CarE genes.These results indicated that the detoxification enzymes of B.tabaci played important roles in response to destruxin A.We then measured the activities of detoxification enzymes and the expression level of some detoxification genes of adults of B.tabaci at 4 hours,8 hours and 12 hours after treated with LC₁₀ and LC₅₀ concentrations of destruxin A.The results showed that when treated with low concentration(LC₁₀)destruxin A,the activities of multi-functional oxidase(MFO),glutathione-S-transferase(GST)and carboxylesterase(CarE) of B.tabaci were significantly increased.The expression of related detoxification genes were up-regulated for detoxification.Under high concentration(LC₅₀)destruxin A,activities of MFO and GST of B.tabaci were significantly inhibited.The qRT-PCR results showed that after the high concentration treatment of destruxin A,cytochrome P450 and GST genes of B.tabaci showed down-regulation.The result further confirmed the inhibitory effect of destruxin A on these two enzymes.The activity of carboxylesterase in B.tabaci had no significant change after 4-hour treatment with destruxin A.However,the activity increased after 8 hours of treatment,with the expression of CarE gene up-regulated.The inhibitory effect of destruxin A on MFO and GST may be the important factors causing the poisoning of B.tabaci.4.The energy substance is the basis for insects to ensure the completion of their own life activities.In order to understand the effect of destruxin A on the energy substances of B.tabaci,the LC₁₀ and LC₅₀ concentrations of destruxin A were measured at 4 hours,8 hours,and 12 hours after treatment.The results showed that the content of trehalose,soluble sugar and glycogen decreased in different degrees after treated with destruxin A,indicating that the energy substances of B.tabaci can be affected.The content of trehalose in B.tabaci after the treatment was significantly decreased,while the activity of trehalase,the specific hydrolase of trehalose,was significantly enhanced.Further studies were analyzed on the expression levels of two genes encoding trehalase,BtTre-1 and BtTre-2 in B.tabaci after treated with destruxin A.We found that destruxin A could induce the expression of the soluble trehalase gene BtTre-1 at both low and high concentrations.However,the expression of the membrane-bound trehalase gene BtTre-2 was not significantly changed.The results of this study can provide experimental data and technical support for excavating the mechanism of destruxins.These results provide a basis for further study for inhibitory and attacking targets of destruxin A against B.tabaci,and for revealing the interaction mechanism between destruxins and insects.