Research On The Defense Mechanism Of Nilaparvata Lugens Under The Stress Of Metarhizium Flavoviride

Nilaparvata lugens St(?)l is a major migratory pest in rice-growing regions of Asia.Metarhizium spp.belong to the Hyphomycetes class of Deuteromycotina subdivision,and it is a type of asexual fungi widely used in prevention and control of agricultural pests.Metarhizium spp.can penetrate the body wall of an insect and kill the host by infecting it.Therefore,Metarhizium spp.exert a positive effect on the host,preventing and ridding the host of piercing and sucking pests.In this study,scanning electron microscopy(SEM)and semi-thin section observation were used to study the process of Metarhizium flavoviride infect N.lugens.Using the ITS region sequence of Metarhizium flavoviride as probe,Fluorescence in situ hybridization(FISH)was used to determine whether Metarhizium flavoviride could infect host's ovaries and transmit to the next generation.To identify the network of regulatory genes involved in the immune response of N.lugens infection with M.flavoviridae,and to further investigate the immune defense mechanism of N.lugens to M.flavoviridae.An Illumina high-throughput sequencing platform was used to analyze the transcriptomes of infected and uninfected N.lugens 24,48 and 72 h after treatment.High-throughput sequencing technology was used to detect changes in the community of symbiotic microorganisms such as bacteria and fungi in N.lugens at different time points after they were sprayed with solutions containing various concentrations of Metarhizium.The aim of this study was to clarify the effects of infection by M.flavoviridae on the structure of symbiotic microbial communities and dominance of certain bacteria in N.lugens.The main results are as follows:(1)Metarhizium spp.easily produces appressorium from the internode membranes and folds,grow hyphae and spore on the body surface,invade host's hemocoel,rapid proliferation and causing host death.M.flavoviridae could not infect and colonize rice tissues,so could not infect egg produced in rice leaf sheath,while it could reproduce on the surface of the isolated eggs.The eggs incubation rate of population treated by M.flavoviridae didn't decrease with the prolongation of treatment time,and there was no significant difference between the duration of nymphs and adults of different treatments.(2)The results of Fluorescent In Situ Hybridization showed that M.flavoviridae detected in the nymphs of the next generation,while whether it could be vertical transmission through ovary and testis needs further tests.(3)An Illumina high-throughput sequencing platform was used to analyze the transcriptomes of infected and uninfected N.lugens 24,48 and 72 h after M.flavoviride treatment.1641,634,736 differentially expressed genes(DEGs)were obtained from the transcriptome data of CK?24 vs.MF?24?CK48 vs.MF48?CK72 vs.MF72.808,253 and 179DEGs were significantly differentially enriched in 94,63 and 63 GO terms,respectively.KEGG pathway analysis indicated that 98,333,and 247 expressed genes were significant differentially enriched in 10,27,and 25 metabolic pathways,respectively.DEGs are mainly enriched in related pathways,such as energy metabolism,ion transport,transcription and translation regulation,reproduction and development regulation,and immune defence response,and most encode potential genes related to immune recognition and regulation.(4)The contents of detoxification enzymes,protective enzymes and phenoloxidase of were all changed after treatment with M.flavoviridae solution,the results showed that the contents of Car E and GST in M.flavoviride-treated N.lugnes population were significantly lower than those in untreated N.lugens at 72h after spraying.CAT and SOD contents in N.lugens were increasing over time after M.flavoviride spraying.The contents of SOD in all 3treated populations were significantly higher than those in untreated populations after 72 h.The contents of PO were not significantly different among treatments at 24 h and 48 h after treatment.However,at 72 h after spraying with 1.0×10~9 spores/L M.flavoviride,the contents of PO in M.flavoviride-treated population increased to 316.43 U/mg,which was significantly higher than those in control population.The invasion of M.flavoviridae results in the decrease of the number of symbiotic yeasts and accumulate of uric acid in the treated population,although it has no effect on the content of essential amino acids in treatment period.However,the lack of raw materials for amino acid synthesis will inevitably make the host unable to get nutritional supplement.(5)In this work,M.flavoviride resulted changes in the structure of the midgut microbial flora,decline of the microbial richness and diversity index.Under the stress of pathogenic fungi,the dominance of bacteria with specific functions in intestinal microorganisms increased.For example,the Proportion of male-killing bacteria,Arsenophonus,and Acinetibacter,Stenotrophomonas,Sphingobacterium,which are related to degradation of exogenous chemicals,are higher than the control population.The research also showed that the N.lugens carried Metarhizium spp.by itself.Although the number of fungal microorganisms in the midgut was relatively simple compared with bacterial microorganisms,some fungal species detected,such as Talaromyces,which produced metabolites that inhibited the activity of DNA polymerase from eukaryotic organisms,were likely to resist exogenous M.flavoviride in the host.