Prophenoloxidase-Mediated Ex Vivo Immunity to Delay Fungal Infection after Insect Ecdysis

Abstract

Skin immunity protects animals from airborne pathogen infection. Unlike mammals, arthropods, including insects, undergo periodic ecdysis to grow and develop. Newly molted insects emerge with unsclerotized thin cuticles but successfully escape pathogenic infections during the post-molt period. Here we show that prophenoloxidases (PPOs) in molting fluids remain bioactive on the integument and impede fungal infection after ecdysis. We found that the purified plasma PPOs or recombinant PPOs could effectively bind to fungal spores (conidia) by targeting the cell wall components chitin and β-1,3-glucan. Pretreatment of the spores of the fungal pathogen Beauveria bassiana with PPOs increased spore hydrophilicity and reduced spore adhesion activity, resulting in a significant decrease in virulence as compared with mock infection. We also identified a spore-secreted protease BPS8, a member of peptidase S8 family of protease that degrade PPOs at high levels to benefit fungal infection, but which at lower doses activate PPOs to inhibit spore germination after melanization. These data indicate that insects have evolved a distinct strategy of ex vivo immunity to survive pathogen infections after ecdysis using PPOs in molting fluids retained on the underdeveloped and tender integument of newly molted insects for protection against airborne fungal infection.

Effect of the insect phenoloxidase on the metabolism of l‐DOPA

“…Exogenous proteases (e.g., α‐chymotrypsin and BPS8) can activate insect PPO (Lu et al., ; Zhang et al., ). But PPO can be degraded if incubation is prolonged (Zhang et al., ).…”

“…Exogenous proteases (e.g., α‐chymotrypsin and BPS8) can activate insect PPO (Lu et al., ; Zhang et al., ). But PPO can be degraded if incubation is prolonged (Zhang et al., ). The low PO activities and low levels of intermediates produced during melanization suggest that exogenous proteases are not efficient inducers of melanization.…”

“…PPO in the molting fluids inhibits bacterial growth and infection by entomopathogenic fungi during the molting. Furthermore, PPO in the molting fluids that remain on the cuticles after ecdysis binds to Beauveria bassiana spores, inhibiting their germination and thereby preventing infection (Zhang et al., ). Therefore, insect PPOs have multiple functions.…”

“…Insect PPOs can be activated by endogenous serine proteases (Ashida & Brey, ; Lu et al., ), protease produced by pathogens (Zhang et al., ), and various compounds (e.g., ethanol and cetylpyridinium chloride (CPC)) (Ashida & Brey, ; Lu et al., ). Although it is still arguable whether ethanol can be utilized to activate PPO in vivo , ethanol is produced by cells under low‐oxygen conditions (John & Greenway, ; Panchal & Stewart, ).…”

Analysis of the functions of the signal peptidase complex in the midgut of Tribolium castaneum

“…After knockdown of TcSPC18 , melanotic masses appeared in the larval and adult midguts (Figure e, Figure o, Figure d and f), which may damage the structure of midgut microvilli and peritrophic membranes. Prophenoloxidase is the direction enzyme to induce melanization in insects (Lu et al., ; Zhang et al., ). However, we do not know whether it had close relationship with this enzyme.…”