The Role Of Prophenoloxidases In The Immune Responses Of The Pea Aphid,Acyrthosiphon Pisum

Aphids are among the most destructive pests in agricultural production.Pea aphid,as a model for studying aphids,was also the first hemimetabolous insect whose genome was sequenced.Compared with other insect species,the pea aphid has an incomplete immune system.Some immune related genes conserved in other insects are missing in the pea aphid genome.Only limited number of immune genes were expressed when the pea aphids were mechanically damaged,infected by microorganisms or parasites.Phenoloxidases(PO)belong to the type 3 copper protein family,and catalyze the hydroxylation of monophenols and the oxidation of the derived o-diphenols,resulting in the formation of o-quinones,which further polymerize to form melanin.Reactive intermediates generated in PO-catalyzed melanization showed direct inhibitory effects to bacteria,fungi,viruses,and parasitic wasps.PO exists in insects as an inactive form,prophenoloxidase(PPO).To evaluate the contribution of phenoloxidase to the pea aphid defense against microbial infection,in this study we knocked down the expression of PPOs and investigated the PO activity,aphids’ survival,and viability of bacteria and fungus in the aphid after infection.The main results are as follows:(1)Knockdown of the PPO1 and PPO2 genes of pea aphids by RNA interference resulted in simultaneous silencing of PPO1 and PPO2 and the silencing of PPO2,respectively.But the expression of PPO1 had no significant decrease after injection of dsPPO1.(2)We examined spontaneous melanization of the hemolymph from the aphids,and found that the knockdown of the PPO expression impedes hemolymph melanization in pea aphids.(3)We next measured PO activity in the hemolymph from PPO-knockdown aphids when they were challenged by P.aeruginosa,S.aureus and B.bassiana.The results showed that neither bacterial infection nor fungal infection affected PO activity in pea aphids.Knockdown of the PPO expression caused a decrease in PO activity in both unchallenged and challenged hemolymph samples.(4)In bacterial and fungal infections,we observed that the knockdown of PPO resulted in more live bacterial cells and fungal spores in the body of the aphids and higher mortality ofthe aphids after infection.This showed that phenoloxidases can resist the invasion of pea aphids by microorganisms,especially Gram-positive bacterium S.aureus and fungus B.bassiana.(5)We found that simultaneous silencing of PPO1 and PPO2 were more susceptible to bacterial or fungal infection than specificity silencing of PPO2,implying that PPO1 and PPO2 are both involved in pea aphid defense against bacteria and fungus.In conclusion,knockdown of PPO genes blocks spontaneous melanization of hemolymph of the pea aphids.Phenoloxidases are involved in pea aphid defense against microbial infection.Our study provided evidence supporting a critical role of PPO in the immune defense of the pea aphid.