Toll signal pathway activating eicosanoid biosynthesis shares its conserved upstream recognition components in a lepidopteran Spodoptera exigua upon infection by Metarhizium rileyi, an entomopathogenic fungus.

Eicosanoids play crucial roles in mediating immune responses in insects. Upon a fungal infection, Toll signal pathway can mediate immune responses of Spodoptera exigua, a lepidopteran insect, by activating eicosanoid biosynthesis. However, upstream signal components of the Toll signal pathway activating eicosanoid biosynthesis remain unclear. This study predicted pattern recognition receptors (PRRs) and serine proteases (SPs) as upstream components of the Toll pathway with reference to known signal components of Manduca sexta, another lepidopteran insect. S. exigua infected with Metarhizium rileyi, an entomopathogenic fungus, activated phospholipase A2 (PLA2) and phenoloxidase (PO) enzymes along with marked increases of expression levels of genes encoding three specific antimicrobial peptides, cecropin, gallerimycin, and hemolin. Among ten Toll receptors encoded in the genome of S. exigua, seven Toll genes were associated with immune responses against fungal infection by M. rileyi through individual RNA interference (RNAi) screening. In addition, two Spätzles (ligands of Toll receptor) were required for Toll signaling against the fungal infection. All predicted upstream components of the Toll pathway were inducible by the fungal infection. Individual RNAi screening showed that three PRRs (βGRP-1, βGRP-2, and GNBP3) and five SPs (ModSP, HP21, HP5, HP6, and HP8) were required for immune responses of S. exigua mediated by Toll signal pathway against the fungal infection. However, two PO-activating proteases (PAP1 and PAP3) were not required for PLA2 activation, although they were required for PO activation. These results suggest that PRRs and SPs conserved as upstream components in Toll signal pathway play crucial roles in triggering eicosanoid biosynthesis of S. exigua to mediate various immune responses against fungal infection.