| Entomophthoralean fungi can cause epidemics in aphid population and play an important role in aphid natural control.Pandora neoaphidis is the most widely distributed aphid specific pathogenic fungus in the world.In previous studies,a gram-negative endofungal bacterium,Acinetobacter calcoaceticus,could harbor into the mycelium of P.neoaphidis,and it had been confirmed by fluorescence in situ hybridization and through the Kohler’s postulates.In addition,A.calcoaceticus could spread from intestine to lipid droplets and survive while fed into the aphid.However,the role of endofungul bacteria in the immune interaction between P.neoaphidis and the aphid is unclear.The genome sequencing showed that aphids lacked the core immune components to deal with gram-negative bacteria.Based on the special immune background of aphids,the immune response of pea aphids to the infection of endofungal bacteria of P.neoaphidis was explored in this study.The results are as followed.1.Screening the pea aphid type and their hemocytes observationDifferent types pea aphid from different regions were identified by diagnostic PCR.The genomic DNA of pea aphid was used as template and the template DNA was amplified by PCR with six specific primers of symbiotic bacteria.The target bands were sequenced for identifying their resource.The aphid type with only one type secondary symbiont was named as LL1 S and was selected to subsequent experiments and.Hemolymph of this pea aphid was extracted and several major hemocytes were observed.2.The survival rate and phendoxidase(PO)activity of pea aphid after injection of two gram-negative bacteriaThe infection experiment of pea aphid of LL1 S strain was carried out by artificial injection.Through the detection of the survival rate and PO activity index of the aphid,Results demonstrated that the survival rate of pea aphid injected with Pseudomonas synxantha(abbr.PS)was significantly lower than that of A.calcoaceticus(abbr.AC).All the aphids died within 48 hours,but the survival rate of the aphids injected with A.calcoaceticus decreased rapidly after 72 hours.PO activity of hemocytes fragments of pea aphid was also measured.Compared with 0 h after injection of P.synxantha,PO activity increased significantly(P < 0.05)in 12 h.Meanwhile,PO activity of hemolymph of aphid injected with A.calcoaceticus have not significant change in0-12 h.However,there was no significant difference in PO activity in the lysate of hemocytes of pea aphid after injection of two gram-negative bacteria,and both of them were at a low level.3.The survival rate and PO activity of pea aphid after fed with two gram-negative bacteriaThe results showed that there was no significant decrease in the survival rate of pea aphid after feeding P.synxantha and A.calcoaceticus,while the activity of PO increased significantly at 12 hours after feeding P.synxantha.According to the results of the two infection ways,P.synxantha may have much more powerful infection to the pea aphid than A.calcoaceticus,and it can rapidly proliferate and spread into the pea aphid.It might activate the phenoloxidase system and cause the change of the PO activity in the hemolymph of the pea aphid during inoculation,while the invasion of A.calcoaceticus may not.4.Transcriptome analysis of pea aphid after injection of two gram-negative bacteriaRNA-seq technology was used to sequence and analyze the pea aphid injected with P.synxantha(PS)and A.calcoaceticus(AC).Negative control was PBS-injected and the genome sequence of A.pisum was used as reference sequence.Between PBS vs AC treatments,2471 DEGs were enriched in 44 GO terms,notably including metabolic process,cell process,macromolecular biosynthesis process,cell,cytoplasmic,structural and molecular activity.Furthermore,2272 DEGs were annotated into 44 KEGG pathways,of which 1167 were up regulated and 1105 were down regulated.The down-regulated pathways include Toll / IMD,MAPK and endocytosis.Between PBS vs PS treatments,a total of 762 DEGs were enriched in 37 GO terms,notably including metabolic process,cell process,stimulation response,signal transduction,membrane part,catalytic activity,etc.1311 DEGs were annotated into 44 KEGG pathways,of which 588 were up-regulated and 723 were down-regulated.The up-regulated DEG mainly focused on thermogenic pathway,proteasome pathway,oxidative phosphorylation pathway,etc.The down regulated DEG mainly focused on Toll / IMD signaling pathway,MAPK signaling pathway,cell adhesion molecule pathway,etc.5.Screening of DEGs related to immune response of pea aphid in different treatment groupsBetween PBS vs AC treatments,94 DEGs were screened,including cathepsin family,serine protease family,dioxygenase,superoxide dismutase,peroxidase reductase,C-type lectin,heat shock protein,glutathione transferase,macrophage migration inhibitor,etc.However,32 immune related DEGs were obtained in PBS vs PS.Different from PBS vs AC,an up-regulated expression of PPO1 was found.In order to explore the co immune related DEGs caused by two kinds of bacteria,94 immune related DEGs of PBS vs AC were compared with 32 immune related DEGs of PBS vs PS.Additionally,27 co-expressed DEGs were found mainly involving serine protease family,cathepsin family and related genes in Toll pathway.Furthermore,in order to compare the DEGs of PS and AC,we also analyzed the DEGs of AC vs PS,screened 51 immune related DEGs.Compared with 27co-expressed DEGs,eight immune related genes whose expression were significantly different were obtained,including 4 up-regulated genes and 4 down regulated genes.These eight genes represented different immune differential genes in PS group and AC group,which are different from those in AC group.Conclusively,injection and feeding with two gram-negative bacteria including P.xanthomonas(PS)and A.calcoaceticus(AC),the pea aphid LL1 S type was explored in this study such as the physiological and biochemical levels(PO activity),transcriptome analysis as well.The obtained results initially verified our speculation that the aphid immune system has a certain "immunity exemption" for gram-negative bacteria.Therefore,this study might help to explain the reason how endofungal bacteria affect the pathogenicity of P.neoaphidis,and also to provide theoretical basis for the mechanism of endofungal bacteria escaping host aphid immune response and for the starting point of designing new pest control strategies. |
