| Lymantria dispar is one of the most destructive leaf eating pests in the world.It feeds on the leaves of more than 300 kinds of coniferous and deciduous forests,seriously harming the healthy development of forests.The use of entomopathogenic fungi,such as Beauveria bassiana,to control the outbreak of agricultural and forestry pests has been widely concerned and applied.The innate immunity of insects and other invertebrates has possessed an important defense mechanism against pathogen invasion in the long-term biological evolution process.However,there is a lack of research on the innate immunity of L.dispar,especially the molecular regulation mechanism between pathogen and host.In addition,the role of gut bacteria,an essential component of host insects,in the biological process of fungal infection is largely unknown.In this study,the stress model of L.dispar to B.bassiana was firstly established.Then,thebasic response characteristics of L.dispar hemocytes and fat bodies under B.bassiana stress were revealed by the second-generation RNA-seq technology and bioinformatics methods,and the immune gene regulatory network of L.dispar was successfully constructed.On this basis,The induced expression patterns and functional verification of antifungal immune elements in L.dispar were compared and analyzed.Finally,the structural and functional changes of gut bacteria after fungal stimulation were analyzed and verified by using 16S r RNA sequencing technology and constructing sterile insect system.The main research contents and results are as follows:(1)The pathogenicities of B.bassiana to L.dispar were determined by blood injection.When conidia concentration was lower than 1×10~5 Condia/m L,B.bassiana showed very low virulence,and there was no significant difference in survival rate between Tween 80 and B.bassiana treated group.The mortality rate of 1×10~6condia/m L conidia was 50%at 6 days and nearly 80%at 8days.The mortality rate of 1×10~7 condia/m L conidia was close to 40%after 3 days of infection and over 90%after 5 days of infection.It was observed that the body weight gain of L.dispar larvae almost stopped after infection with B.bassiana(P<0.05),α-amylase and lipase activities in midgut of larvae were significantly decreased(P<0.05).At the initial stage of fungal infection(24h),the activity of superoxide dismutase(SOD)increased(P<0.01),malondialdehyde content increased significantly(P<0.001);The relative content of glutathione(GSH)decreased and the relative activity of superoxide dismutase(SOD)was inhibited in the middle and late period of fungal infection(48,72h).The total antioxidant capacity was significantly down-regulated(P<0.001).These results indicated that B.bassiana was highly pathogenic to L.dispar,causing digestive disorder in midgut and significantly inhibiting the larval defense system.(2)The second generation RNA-Seq technique was used to detect the gene expression patterns of hemocytes and fat bodies of L.dispar infected by B.bassiana.A total of 63235 Unigenes were obtained,with an average length of 965.04bp and an N50 length of 1501bp.Hemocytes and fat body of differentially expressed genes of 2078 and 3465 respectively(|log2 fold change|>1),599 genes were co-expressed in blood cells and adiposomes.GO analysis showed that these genes were mainly enriched in cellular process,metabolic process,catalytic activity and binding.KEGG pathway analysis showed that the differential genes were mainly enriched in Toll and Imd signaling pathways,protein processing signaling pathways and TCA cycle signaling pathways.Hierarchical cluster analysis of differentially expressed genes in hemocytes and fat body showed the same expression pattern in Tween 80 treated larval Hemocytes and fat bodies as controls,while the RNA levels of B.bassiana infected larval hemocytes were significantly higher than those of the control group.(3)A total of 244 genes related to immunity of L.dispar were manually annotated from 63235 Unigenes by BLASTX search and compared with other known protein sequences of other insects.The immunorelated genes of L.dispar were classified into pathogen recognition molecules,extracellular signal regulation,intracellular signal transduction and immune response effector factors according to their functions.L.dispar innate immune system function is completed,and there is no absence of immune elements.In this study,anti-fungal immune Toll pathway and anti-bacterial immune Imd pathway were verified.The results showed that recognition genes(PGRP-S2,CTL3,βGRP4)were significantly upregulated after 48h fungal infection.Signal regulation genes(CSP8,SPH1)were up-regulated or significantly up-regulated in adipocytes and hemocytes.In contrast,signal regulation(Serpin9),Toll pathway(Toll6,SPZ3)and effector factors(Lys4,ATT2)were down-regulated at 72 h after fungal infection.In addition,the expression of all proppo genes in the host blood cells increased at 48 and 72h after fungal infection,and the activity of PO increased significantly at 48 and 72h after fungal infection,suggesting that melanism was a positive response to the immune response of gypsy-moth to B.bassiana.(4)Through artificial isolation,culture and identification,a total of 7 strains of L.dispar gut bacteria were obtained.The results of 16S r RNA sequencing showed that the gut bacteria structure of L.dispar larvae was significantly affected by food.The gut bacteria cultured in the laboratory is simple in structure,including Enterococcus,Thauera,Pseudomonas,Weissella and Lactobacillus.Enterococcus is the dominant bacteria in indoor breeding.However,the gut bacteria of L.dispar in the wild is diverse and complex,and there is no fixed dominant bacteria.At 48h and 72h of fungal infection,the proportion of Rosenbergiella bacteria increased significantly.Principal coordinate analysis showed that there were significant differences in the intestinal microbiota structure between the control group and B.bassiana infected larvae 48 h after infection.The number of gut microbiota in the fungal infected larvae was significantly higher than that in the control group,and the level of ROS in the intestinal tract of the fungal infected larvae was significantly lower than that in the control group.Bioassay results showed that the virulence of B.bassiana decreased and the intestinal flora migrated to the blood lumen,which helped B.bassiana to accelerate the death of the larvae.To sum up,the ball spore beauveria bassiana has extremely strong pathogenicity to the L.dispar,significantly activated L.dispar innate immune response cascade,in the process of the interaction of an arms race,and gut bacteria as a special"multifunctional organ"insects,in the middle and later periods of the fungal infection to participate in the competition,plays a role that nots allow to ignore.This study provides a comprehensive sequence resource and important new insights into the forest pest L.dispar,and elucidates the complex multi-component interaction between host,pathogenic fungi,and intestinal flora,which contributes to better innate immunity of detoxizing moth,thus providing theoretical basis for integrated pest control. |
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