A Nonpermissive Entomophthoralean Fungal Infection Increases Activation of Insect Prophenoloxidase

Abstract

Entomophaga maimaigaandEntomophaga aulicaeare entomopathogenic fungi that show species-specific infection in Lepidoptera. These fungi grow as protoplasts in the hemolymph of permissive insect hosts.E. maimaigainfects gypsy moth larvae,Lymantria dispar,andE. aulicaeinfects hemlock looper,Lambdina fiscellaria.Cross-infections do not occur and are referred to as a nonpermissive response. We circumvented cuticular barriers and injectedE. aulicaeprotoplasts intoL. disparand investigated factors which could potentially curtail entomophthoralean fungal infection in a nonpermissive host insect. There was no evidence of (i) restricted fungal growth in cell-free hemolymph of the nonpermissive host, (ii) fungal toxins specific to host insect, (iii) hemocyte encapsulation of fungal protoplasts in a nonpermissive or permissive infection, or (iv) fungal-specific induction of plasma proteins inL. dispar.However, higher levels of phenoloxidase activity for up to 96 h postchallenge, as well as a prophenoloxidase-activating trypsin activity, were observed forL. disparchallenged withE. aulicaewhen compared to anE. maimaigachallenge. Three isoforms of phenoloxidase (pI5.0–5.5) and at least six isoforms of trypsin activity (four basic trypsins pI8–10 and two acidic trypsins pI4–6) with preferences for small amino acid residues were activated inL. disparafter challenge.In vitroprophenoloxidase activation experiments showed that treatment ofL. disparhemolymph withE. aulicaeprotoplast plasma membranes consistently resulted in higher prophenoloxidase activation thanE. maimaiga.We suggest that differences in protoplastic components ofEntomophagaspecies, such as the surface glycoproteins, are implicated in activation of zymogenic trypsins in the insect which in turn activate the prophenoloxidase cascade as a nonpermissive response.