The Role Of Reactive Oxygen Species And Facultative Symbiont In Pathogen Defenses Of Pea Aphid,Acyrthosiphon Pisum

Pea aphid Acyrthosiphon pisum is the first genomic sequenced Hemiptera insect,and it also harbors one obligate symbiont,Buchnera aphidicola,and several facultative symbionts.This makes pea aphid a valuable model to study the interaction between aphids and microorganisms.To date,it is rarely known about the imunity response to foreign microbes in pea aphid.Reactive oxygen species?ROSs?can be generated by living organisms to fight against foreign microbe invasion.Previous studies have shown that facultative symbionts can protect aphids against parasitic wasps and the fungal pathogen Pandora neoaphidis.In this study,we investigated the levels of H₂O₂ and expressions of ROS-related genes in A.pisum after bacterial infection.The A.pisum Preoxiredoxin 1,2?ApPrx1,ApPrx2?were cloned and their function in pea aphid's immune response to bacteria were characterized.The presence of facultative symbionts in our laboratory pea aphid strain was examinated,and its effect on immune response in aphids was determined.The results were as follows:Both infected with Pseudomonas aeruginosa and Micrococcus luteus caused aphids' significantly death,and the aphids dead much more quickly upon P.aeruginosa infection.The H₂O₂ levels increased significantly in aphids after Pseudomonas aeruginosa and Micrococcus luteus infection.Compare to P.aeruginosa infected aphids,the H₂O₂ levels increased more dramatically in M.luteus infected aphids.Different expressions were found in ROS-related genes,including dual oxidase?DUOX?,superoxide dismutase?SOD?and ROS detoxification enzymes,such as Peroxiredoxins.Recombinant ApPrx1 and ApPrx2 protein from Escherichia coli showed antioxidative activity by eliminating H₂O₂ effectively,and could protect DNA from oxidative damage.ApPrx1 and ApPrx2 expression was remarkably up-regulated when the aphids were infected with bacteria or injected with H₂O₂.Micro-injectoin of dsRNA was used to knockdown of ApPrx1 and ApPrx2.The significant decrease of expression was observed at 3 d after dsRNA injection,and the RNAi efficiency was 35% and 50% on ApPrx1 and ApPrx2,respectively.When ApPrx1 expression was reduced by dsRNA injection,the survival of aphids was significantly decreased after bacteria infection.Knockdown of ApPrx1 decreased bacterialoads inside the aphids post infection.While under infection condition,the H₂O₂ levels were much higher in ApPrx1 knockdown aphids than in dsGFP-injected aphids.When ApPrx2 was knockdown,the similar results were observed,indicating that the declined survival of aphids was caused by increased oxidative stress.The facultative symbionts were determined in our laboratory pea aphid strain,and only Serratia symbiotica was detected.Microinjection of ampicillin cured the aphids and established an S.symbiontica-free aphid strain.Different species of bacteria?Staphylococcus aureus,M.luteus,Streptococcus pneumoniae,P.aeruginosa,Serratia marcescens,and E.coli?were infected the aphids either with or without S.symbiontica.Compared to the wild-type strain,however,no significant difference in survival was observed in the S.symbiontica-free aphid strain.Beauveria bassiana,a common entomopathogenic fungus,was also introduced.However,survival did not differ significantly between the two strains.In addition,the density of S.symbiotica after bacterial infection increased remarkably?approximately 2-fold to 3-fold?following infection of different bacteria.The density of B.aphidicola was also examined after bacterial infection.Interestingly,B.aphidicola significantly increased only upon infection with Gram-positive bacteria,particularly M.luteus.On the contrary,infection with B.bassiana did not change the densities of either S.symbiotica or B.aphidicola.These results indicate that S.symbiotica does not affect the survival of A.pisum upon bacterial or fungal infection.Taken together,we preliminarily demonstrated that ROS involved in the defense of pea aphid to bacteria,and Prxs play a crucial role in regulation of ROS homeostasis inside aphids under infection.Faclutative symbiont S.symbiotica does not protect the aphids from bacterial or Beauveria bassiana infection,however,bacterial infection can cause the multiplying of S.symbiotica and B.aphidicola.