Biochemical And Molecular Mechanisms Of Btrice Affecting The Population Dynamics Of Parasitoid In Paddy Fields

The application of insect-resistant genetically engineering(IRGE)crops effectively control target insect pests,reduce the use of chemical insecticides and protect human health and ecological balance.In order to control rice lepidopteran insect pests,Chinese scientists have developed dozens of IRGE rice lines expressing insecticidal proteins from the soil bacterium Bacillus thuringiensis(Bt).Laboratory and field experiments have shown that the narrow target insect spectrum and high specifity of Bt protein make it poses no direct toxic effects on parasitoids.However,field surveys found that parasitoids of target and non-target pests were significantly lower in Bt than that in non-Bt rice fields.The ecological mechanism of this phenomena is still unknown.In this study,we conducted series biological,ecological,molecular and omics experiments based on tritrophic systems including Bt/non-Bt rice,Bt rice target insect pest Chilo suppressalis and its larval parasitoids Cotesia chilonis,Bt rice non-target insect pest brown planthopper Nilaparvata lugens and its egg parasitoid Anagrus nilaoarvatae to explore the effects of plantation of Bt rice on the population density of parasitoid in fields.The molecular and biochemical mechanisms for explaining the potential effects were elucidated.The mainly results were summarized as follows:(1)Y-tube olfactometer studies revealed that C.chilonis females responded similarly to undamaged Bt and non-Bt rice plants.Parasitoids preferred rice plants damaged by 3rd-instar larvae of C.suppressalis,but did not differentiate between caterpillar-infested Bt and non-Bt plants.According to GC-MS analyses of rice plant volatiles,undamaged Bt and non-Bt rice plants emitted the same number of volatile compounds and there were no significant differences in the quantity of each and total volatile compounds between the treatments.When plants were infested with and damaged by C.suppressalis larvae,both Bt and non-Bt rice plants emitted higher numbers and larger amounts of volatile compounds than undamaged plants,but there were no significant differences between Bt and non-Bt plants.These results suggest that genetic engineering of the Bt rice has not altered the secondary metabolism of rice with respect to volatile synthesis/emissions.While when Bt rice and non-Bt rice were adjacently grown,the target pest C.suppressalis caused much heavier damage on non-Bt rice plants than on Bt rice plants,resulting that non-Bt rice plants released significantly larger amounts of volatile compounds compared to Bt rice plants.Thus,non-Bt rice plants becomed more attractive to C.chilonis than Bt rice,consequently causing a significant higher population density of C.chilonis in non-Bt rice fields.(2)Behavior experiments of N.lugens showed that female adults significantly preferred feeding and ovipositing on caterpillar-free non-Bt rice plants than Bt plants.Moreover,female adults preferred feeding and laied more eggs on non-Bt plants infested with 3rd-instar C.suppressalis larvae than caterpillar-free non-Bt or Bt rice plants.Y-tube olfactometer studies showed that A.nilaoarvatae females did not exhibit a significantly different preference for rice plants that were damaged by1st-instar C.suppressalis larvae or undamaged plants.In contrast,rice plants infested with female N.lugens adults were significantly more attractive to A.nilaoarvatae than undamaged plants.These results indicated that female N.lugens preferred feeding and ovipositing on rice plants infested by C.suppressalis.We inferred that non-Bt rice plants were more damaged by C.suppressalis in paddy fields,and were more attractive to N.lugens adults,finally resulting in high population density of N.lugens in non-Bt rice fields.The population density of A.nilaoarvatae in rice field is a chain reaction result from its host N.lugens dispersal from Bt to non-Bt rice fields,consequently causing a similar significant higher population density of A.nilaoarvatae in non-Bt rice fields.(3)Based on above results,we concluded that the host choice behavior of parasitoids in paddy fields were not directly related to the genetic background of rice lines,but were results brought by the different infestation level of target pest C.suppressalis.Thus we conducted experiments to reveal potential biochemical and molecular mechanism of Bt rice affect the population dynamic of parasitoid in paddy fields.We combined next-generation RNA sequencing and metabolomics techniques to investigate the dynamic changes in gene expression and in metabolic processes in rice plants that had been continuously fed by C.suppressalis larvae for different durations(0,24,48,72,and 96 h).Furthermore,the data were validated using quantitative real-time PCR.Transcriptome analyses showed that a total of 4729 differentially expressed genes(DEGs)were detected at a minimum of two time points,and these genes can be divided into five groups according their expression dynamics.GO and KEGG pathway analyses showed that these DEGs involved in a lot of primary and secondary metabolisms.A total of 151 known metabolites were detected by metabolome analyses.we divided the metabolites into seven classes including amino acids,carbohydrates,lipids,cofactors,prosthetic groups,and electron carriers,nucleotides,peptides and secondary metabolites.Among these metabolites,the contents of most amino acids,especially asparagine,which is serving as sucking stimulants for N.lugens and total amino acids were increased significantly.Moreover,N.lugens sucking-inhibitory chemicals such as β-sitosterol,and campesterol were significantly decreased compared to uninfested rice plants.In addition,volatiles released by C.suppressalis larvae infested rice plants were attractive to N.lugens((E)-β-caryophyllene,2-heptanol,α-pinene,and limonene)and C.chilonis(limonene and2-nonanone).Further combined omics data revealed systemic and dynamic processes of rice plants to C.suppressalis attack,which involving genes and metabolites regarding primary metabolisms including photosynthesis,amino acid metabolism,and carbohydrate metabolism,and secondary metabolisms such as the biosynthesis of phenylpropanoids and terpenoids.On the basis of these results,we concluded that the changes in metabolic processes of rice infested with C.suppressalis were the main cause for uneven distribution of N.lugens and parasitoid in rice paddy fields.In this study,the molecular and biochemical mechanisms for explaining the potential effects of planting of Bt rice on the population dynamic of the parasitoids of target pests were expounded.It also provided us a scientific evidence for better understanding of the impacts of insect-resistant genetically modified crops on the biodiversity,the evolution of resistance of target pests and the mechanisms of the outbreak of secondary pests in Bt crop fields.